Method for screening nucleic acid catalysts

ABSTRACT

Nucleic acid catalysts, method of screening for variants of nucleic acid catalysts, synthesis of ribozyme libraries and discovery of gene sequences are described.

[0001] This patent application claims priority to U.S. patentapplication Ser. No. 60/068,212 “METHOD FOR SCREENING NUCLEIC ACIDCATALYSTS”, Burgin et al., filed Dec. 19, 1997, and is a CIP of U.S.patent application Ser. No. 09/094,38 “METHOD FOR SCREENING NUCLEIC ACIDCATALYSTS”, Burgin et al., filed Jun. 9, 1998 which claims priority toU.S. patent application Ser. No. 60/049,002, filed Jun. 9, 1997.

BACKGROUND OF THE INVENTION

[0002] This invention relates to nucleic acid molecules with catalyticactivity and derivatives thereof.

[0003] The following is a brief description of catalytic nucleic acidmolecules. This summary is not meant to be complete but is provided onlyfor understanding of the invention that follows. This summary is not anadmission that all of the work described below is prior art to theclaimed invention.

[0004] Catalytic nucleic acid molecules (ribozymes) are nucleic acidmolecules capable of catalyzing one or more of a variety of reactions,including the ability to repeatedly cleave other separate nucleic acidmolecules in a nucleotide base sequence-specific manner. Such enzymaticnucleic acid molecules can be used, for example, to target virtually anyRNA transcript (Zaug et al., 324, Nature 429 1986; Cech, 260 JAMA 3030,1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989). Anynucleotide base-comprising molecule having the ability to repeatedly acton one or more types of molecules, including but not limited toenzymatic nucleic acid molecules. By way of example but not limitation,such molecules include those that are able to repeatedly cleave nucleicacid molecules, peptides, or other polymers, and those that are able tocause the polymerization of such nucleic acids and other polymers.Specifically, such molecules include ribozymes, DNAzymes, external guidesequences and the like. It is expected that such molecules will alsoinclude modified nucleotides compared to standard nucleotides found inDNA and RNA

[0005] Because of their sequence-specificity, trans-cleaving enzymaticnucleic acid molecules show promise as therapeutic agents for humandisease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294;Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymaticnucleic acid molecules can be designed to cleave specific RNA targetswithin the background of cellular RNA. Such a cleavage event renders themRNA non-functional and abrogates protein expression from that RNA. Inthis manner, synthesis of a protein associated with a disease state canbe selectively inhibited.

[0006] There are at least seven basic varieties of naturally-occurringenzymatic RNAs. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

[0007] In addition, several in vitro selection (evolution) strategies(Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker etal., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418;Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183;Breaker, 1996, Curr. Op. Biotech., 7, 442; Breaker, 1997, NatureBiotech. 15, 427).

[0008] There are several reports that describe the use of a variety ofin vitro and in vivo selection strategies to study structure andfunction of catalytic nucleic acid molecules (Campbell et al., 1995, RNA1, 598; Joyce 1989, Gene, 82,83; Lieber et al., 1995, Mol Cell Biol. 15,540; Lieber et al., International PCT Publication No. WO 96/01314;Szostak 1988, in Redesigning the Molecules of Life, Ed. S. A. Benner, pp87, Springer-Verlag, Germany; Kramer et al., U.S. Pat. No. 5,616,459;Joyce, U.S. Pat. No. 5,595,873; Szostak et al., U.S. Pat. No.5,631,146).

[0009] The enzymatic nature of a ribozyme is advantageous over othertechnologies, since the effective concentration of ribozyme necessary toeffect a therapeutic treatment is generally lower than that of anantisense oligonucleotide. This advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme (enzymaticnucleic acid) molecule is able to cleave many molecules of target RNA.In addition, the ribozyme is a highly specific inhibitor, with thespecificity of inhibition depending not only on the base-pairingmechanism of binding, but also on the mechanism by which the moleculeinhibits the expression of the RNA to which it binds. That is, theinhibition is caused by cleavage of the RNA target and so specificity isdefined as the ratio of the rate of cleavage of the targeted RNA overthe rate of cleavage of non-targeted RNA. This cleavage mechanism isdependent upon factors additional to those involved in base-pairing.Thus, it is thought that the specificity of action of a ribozyme isgreater than that of antisense oligonucleotide binding the same RNAsite.

[0010] The development of ribozymes that are optimal for catalyticactivity would contribute significantly to any strategy that employsRNA-cleaving ribozymes for the purpose of regulating gene expression.The hammerhead ribozyme functions with a catalytic rate (k_(cat)) of ˜1min⁻¹ in the presence of saturating (10 mM) concentrations of Mg²⁺cofactor. However, the rate for this ribozyme in Mg²⁺ concentrationsthat are closer to those found inside cells (0.5-2 mM) can be 10- to100-fold slower. In contrast, the RNase P holoenzyme can catalyzepre-tRNA cleavage with a k_(cat) of ˜30 min⁻¹ under optimal assayconditions. An artificial ‘RNA ligase’ ribozyme (Bartel et al., supra)has been shown to catalyze the corresponding self-modification reactionwith a rate of ˜100 min⁻¹. In addition, it is known that certainmodified hammerhead ribozymes that have substrate binding arms made ofDNA catalyze RNA cleavage with multiple turnover rates that approach 100min⁻¹. Finally, replacement of a specific residue within the catalyticcore of the hammerhead with certain nucleotide analogues gives modifiedribozymes that show as much as a 10-fold improvement in catalytic rate.These findings demonstrate that ribozymes can promote chemicaltransformations with catalytic rates that are significantly greater thanthose displayed in vitro by most natural self-cleaving ribozymes. It isthen possible that the structures of certain self-cleaving ribozymes maynot be optimized to give maximal catalytic activity, or that entirelynew RNA motifs could be made that display significantly faster rates forRNA phosphoester cleavage.

[0011] An extensive array of site-directed mutagenesis studies have beenconducted with ribozymes such as the hammerhead, hairpin, hepatitisdelta virus, group I, group II and others, to probe relationshipsbetween nucleotide sequence, chemical composition and catalyticactivity. These systematic studies have made clear that most nucleotidesin the conserved core of these ribozymes cannot be mutated withoutsignificant loss of catalytic activity. In contrast, a combinatorialstrategy that simultaneously screens a large pool of mutagenizedribozymes for RNAs that retain catalytic activity could be used moreefficiently to define immutable sequences and to identify new ribozymevariants.

[0012] Although in vitro selection experiments have been reported withthe hammerhead ribozyme (Nakamaye & Eckstein, 1994, Biochemistry 33,1271; Long & Uhlenbeck, 1994, Proc. Natl. Acad. Sci., 91, 6977; Ishizakaet al., 1995, BBRC 214, 403; Vaish et al., 1997, Biochemistry, 36, 6495)and Hairpin ribozyme (Berzal et al., 1993, EMBO, J., 12, 2567) none ofthese efforts have successfully screened for all possible combinationsof sequence and chemical variants that encompass the entire catalyticcore.

[0013] The references cited above are distinct from the presentlyclaimed invention since they do not disclose and/or contemplate theenzymatic nucleic acid molecules and the methods for screening ribozymevariants.

SUMMARY OF THE INVENTION

[0014] This invention relates to novel nucleic acid molecules withcatalytic activity, which are particularly useful for cleavage of RNA orDNA. The nucleic acid catalysts of the instant invention are distinctfrom other nucleic acid catalysts known in the art. This invention alsorelates to a method of screening variants of nucleic acid catalystsusing standard nucleotides or modified nucleotides. Applicant hasdetermined an efficient method for screening libraries of catalyticnucleic acid molecules, particularly those with chemical modificationsat one or more positions. The method described in this applicationinvolves systematic screening of a library or pool of ribozymes withvarious substitutions at one or more positions and selecting forribozymes with desired function or characteristic or attribute.

[0015] Applicant describes herein, a general combinatorial approach forassaying ribozyme variants based on ribozyme activity and/or a specific“attribute” of a ribozyme, such as the cleavage rate, cellular efficacy,stability, delivery, localization and the like. Variations of thisapproach also offer the potential for designing novel catalyticoligonucleotides, identifying ribozyme accessible sites within a target,and for identifying new nucleic acid targets for ribozyme-mediatedmodulation of gene expression.

[0016] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The drawings will first briefly be described.

[0018] Drawings:

[0019]FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain known in the art. Stem II can be 2 base-pair long. Each N isindependently any base or non-nucleotide as used herein.

[0020]FIG. 2a is a diagrammatic representation of the hammerheadribozyme domain known in the art; FIG. 2b is a diagrammaticrepresentation of the hammerhead ribozyme as divided by Uhlenbeck (1987,Nature, 327, 596-600) into a substrate and enzyme portion; FIG. 2c is asimilar diagram showing the hammerhead divided by Haseloff and Gerlach(1988, Nature, 334, 585-591) into two portions; and FIG. 2d is a similardiagram showing the hammerhead divided by Jeffries and Symons (1989,Nucl. Acids. Res., 17, 1371-1371) into two portions.

[0021]FIG. 3 is a diagrammatic representation of the general structureof a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 basepairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally providedof length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20or more). Helix 2 and helix 5 may be covalently linked by one or morebases (i.e., r is 1 base). Helix 1, 4 or 5 may also be extended by 2 ormore base pairs (e.g., 4-20 base pairs) to stabilize the ribozymestructure, and preferably is a protein binding site. In each instance,each N and N′ independently is any normal or modified base and each dashrepresents a potential base-pairing interaction. These nucleotides maybe modified at the sugar, base or phosphate. Complete base-pairing isnot required in the helices, but is preferred. Helix 1 and 4 can be ofany size (i.e., o and p is each independently from 0 to any number,e.g., 20) as long as some base-pairing is maintained. Essential basesare shown as specific bases in the structure, but those in the art willrecognize that one or more may be modified chemically (abasic, base,sugar and/or phosphate modifications) or replaced with another basewithout significant effect. Helix 4 can be formed from two separatemolecules, i.e., without a connecting loop. The connecting loop whenpresent may be a ribonucleotide with or without modifications to itsbase, sugar or phosphate. “q” is 2 bases. The connecting loop can alsobe replaced with a non-nucleotide linker molecule. H refers to bases A,U, or C. Y refers to pyrimidine bases. “______” refers to a covalentbond.

[0022]FIG. 4 is a representation of the general structure of thehepatitis delta virus ribozyme domain known in the art. In eachinstance, each N and N′ independently is any normal or modified base andeach dash represents a potential base-pairing interaction. Thesenucleotides may be modified at the sugar, base or phosphate.

[0023]FIG. 5 is a representation of the general structure of theself-cleaving VS RNA ribozyme domain.

[0024]FIG. 6 is a schematic representation of a combinatorial approachto the screening of ribozyme variants.

[0025]FIG. 7 shows the sequence of a Starting Ribozyme to be used in thescreening approach described in FIG. 6. The Starting Ribozyme is ahammerhead (HH) ribozyme designed to cleave target RNA A (HH-A).Position 7 in HH-A is also referred to in this application as position24 to indicate that U24 is the 24th nucleotide incorporated into theHH-A ribozyme during chemical synthesis. Similarly, positions 4 and 3are also referred to as positions 27 and 28, respectively. s indicatesphosphorothioate substitution. Lower case alphabets in the HH-A sequenceindicate 2′-O-methyl nucleotides; uppercase alphabets in the sequence ofHH-A at positions 5, 6, 8, 12 and 15.1 indicate ribonucleotides.Positions 3, 4 and 7 are shown as uppercase, large alphabets to indicatethe positions selected for screening using the method shown in FIG. 6. indicates base-paired interaction. iB represents abasic inverted deoxyribose moiety.

[0026]FIG. 8 shows a scheme for screening variants of HH-A ribozyme.Positions 24, 27 and 28 are selected for analysis in this scheme.

[0027]FIG. 9 shows non-limiting examples of some of the nucleotideanalogs that can be used to construct ribozyme libraries. 2′-O-MTM-Urepresents 2′-O-methylthiomethyl uridine; 2′-O-MTM-C represents2′-O-methylthiomethyl cytidine; 6-Me-U represents 6-methyl uridine(Beigelman et al., International PCT Publication No. WO 96/18736 whichis incorporated by reference herein).

[0028]FIG. 10 shows activity of HH-A variant ribozymes as determined ina cell-based assay. * indicates the substitution that provided the mostdesirable attribute in a ribozyme.

[0029]FIG. 11A shows the sequence and chemical composition of ribozymesthat showed the most desirable attribute in a cell.

[0030]FIG. 11B shows formulae for four different novel ribozyme motifs.

[0031]FIG. 12 shows the formula foe a novel ribozyme motif.

[0032]FIG. 13 shows the sequence of a Starting Ribozyme to be used inthe screening approach described in FIG. 14. A HH ribozyme targetedagainst RNA B (HH-B) was chosen for analysis of the loop II sequencevariants.

[0033]FIG. 14 shows a scheme for screening loop-II sequence variants ofHH-B ribozyme.

[0034]FIG. 15 shows the relative catalytic rates (k_(rel)) for RNAcleavage reactions catalyzed by HH-B loop-II variant ribozymes.

[0035]FIG. 16 is a schematic representation of HH-B ribozyme-substratecomplex and the activity of HH-B ribozyme with either the 5′-GAAA-3′ orthe 5′-GUUA-3′ loop-II sequence.

[0036]FIG. 17 shows a scheme for using a combinatorial approach toidentify potential ribozyme targets by varying the binding arms.

[0037]FIG. 18 shows a scheme for using a combinatorial approach toidentify novel ribozymes by the varying putative catalytic domainsequence.

[0038]FIG. 19 shows a table of accessible sites within a Bcl-2transcript (975 nucleotides) which were found using the combinatorial invitro screening process.

[0039]FIG. 20 shows a table of accessible sites with a K-ras transcript(796 nucleotides) which were found using the combinatorial in vitroscreening process as well as a graphic depiction of relative activity ofribozymes to those sites. All potential hammerhead ribozyme cleavagesites are indicated in the graph with a short vertical line. The actualsites identified are indicated in the graph. The size of the barreflects the intensity of the cleavage product from the cleavagereaction. The actual sequence of each site, the sequence I.D. number,the position of cleavage within the transcript (based on the knownsequence), and the estimated size of the cleavage product (based on gelanalysis) are listed.

[0040]FIG. 21 shows a table of accessible sites with a urokinaseplasminogen activator (UPA) transcript (400 nucleotides) which werefound using the combinatorial in vitro screening process as well as agraphic depiction of relative activity of ribozymes to those sites. Allpotential hammerhead ribozyme cleavage sites are indicated in the graphwith a short vertical line. The actual sites identified are indicated inthe graph. The size of the bar reflects the intensity of the cleavageproduct from the cleavage reaction. The actual sequence of each site,the sequence I.D. number, the position of cleavage within the transcript(based on the known sequence), and the estimated size of the cleavageproduct (based on gel analysis) are listed.

[0041]FIG. 22 shows a graph displaying data from a ribonucleaseprotection assay (RPA) after treatment of MCF-7 cells with ribozymes totargeted to site 549 of the transcript (Seq.ID No.9). The Bcl-2 mRNAisolated from MCF-7 cells is normalized to GAPDH which was also probedin the RPA. The graph includes an untreated control and an irrelevantribozyme (no complementarity with Bcl-2 mRNA).

[0042]FIG. 23 shows a schematic representation of the ribozymessynthesized to screen for accessible sites within in vitro transcripts.

[0043] In one preferred embodiment, the method relies upon testingmixtures (libraries) of ribozymes with various nucleotides, nucleotideanalogs, or other analog substitutions, rather than individualribozymes, to rapidly identify the nucleotide, nucleotide analog, orother analog that is variable at one or more positions within aribozyme. In the first step (step 1, FIG. 6), a desired number ofpositions (for example, 3 positions as shown in FIG. 6) are chosen forvariation in a first ribozyme motif (Starting Ribozyme); there is norequirement on the number of positions that can be varied and thesepositions may or may not be phylogenetically conserved for the ribozyme.In addition, these position may reside within the catalytic core,binding arms, or accessory domains. The number of positions that arechosen to be varied defines the number of “Classes” of ribozymelibraries that will be synthesized. In the example illustrated in FIG.6, three positions (designated positions 1, 2 and 3) are varied, sothree different Classes of ribozyme pool are synthesized. In the nextstep (step 2), ribozyme pools are synthesized containing a randommixture of different nucleotides, nucleotide analogs, or other analogsat all of the desired positions (designated “X”) to be varied exceptone, which is the “fixed” position (designated “F”). The fixed positioncontains a specific nucleotide, nucleotide analog or other analog. Thereis no requirement for the number of nucleotides, or analogs be used. Thenumber of nucleotides or analogs defines the number of pools (designatedn) in each Class. For example if ten different nucleotides or analogsare chosen, ten different pools (n=10) will be synthesized for eachClass; each of the pools will contain a specific modification at onefixed position (designated F) but will contain an equal mixture of allten modifications at the other positions (designated X). In a subsequentstep (step 3), the different pools of ribozymes are tested for desiredactivity, phenotype, characteristic or attribute. For example, thetesting may be determining in vitro rates of target nucleic acidcleavage for each pool, testing ribozyme-substrate binding affinities,testing nuclease resistance, determining pharmacodynamic properties, ordetermining which pool is most efficacious in a cellular or animal modelsystem. Following testing, a particular pool is identified as a desiredvariant (designated “Desired Variant-1”) and the nucleotide or theanalog present at the fixed position within the Desired Variant-1 ismade constant (designated “Z”) for all subsequent experiments; a singleposition within a ribozyme is therefore varied, i.e., the variablenucleotide or analog at a single position, when all other X positionsare random, is identified within a ribozyme motif. Subsequently, newribozyme pools (Classes 2, 3 etc.) are synthesized containing an equalmixture of all nucleotides or analogs at the remaining positions to beoptimized except one fixed position and one or more constant positions.Again, a specific nucleotide or analog is “fixed” at a single positionthat is not randomized and the pools are assayed for a particularphenotype or attribute (step 4). This process is repeated until alldesired positions have been varied and screened. For example if threepositions are chosen for optimization, the synthesis and testing willneed to be repeated three times (3 Classes). In the first two Classes,pools will be synthesized; in the final Class, specific ribozymes willbe synthesized and tested. When the final position is analyzed (step 5),no random positions will remain and therefore only individual ribozymesare synthesized and tested. The resulting ribozyme or ribozymes(designated “second ribozyme motif”) will have a defined chemicalcomposition which will likely be distinct from the Starting Ribozymemotif (first ribozyme motif). This is a rapid method of screening forvariability of one or more positions within a ribozyme motif.

[0044] In another preferred embodiment, the invention involves screeningof chemical modifications at one or more positions within a hammerheadribozyme motif. More specifically, the invention involves variability inthe catalytic core sequence of a hammerhead ribozyme. Particularly, theinvention describes screening for variability of positions 3, 4 and 7within a hammerhead ribozyme. The invention also features screening foroptimal loop II sequence in a hammerhead ribozyme.

[0045] In yet another preferred embodiment, the invention features arapid method for screening accessible ribozyme cleavage sites within atarget sequence. This method involves screening of all possiblesequences in the binding arm of a ribozyme. The sequence of the bindingarms determines the site of action of certain ribozymes. Thecombinatorial approach can be used to identify desirable and/oraccessible sites within a target sequence by essentially testing allpossible arm sequences. The difficulty with this approach is thatribozymes require a certain number of base pairs (for example, forhammerhead ribozymes the binding arm length is approximately 12-16nucleotides) in order to bind functionally and sequence-specifically.This would require, for example 12-16 different groups of hammerheadribozyme pools; 12-16 positions would have to be optimized which wouldrequire 12-16 different groups being synthesized and tested. Each poolwould contain the four different nucleotides (A, C, U and G) ornucleotide analogs (p=4 for nucleotides). It would be very timeconsuming to test each group, identify the best pool, synthesize anothergroup of ribozyme pools with one additional position constant, and thenrepeat the procedure until all 12-16 groups had been tested. However itis possible to decrease the number of Classes by testing multiplepositions within a single Class. In this case, the number of poolswithin a Class equals the number of nucleotides or analogs in the randommixture (i.e. n) to the w power, where w equals the number of positionsfixed in each Class. The number of Classes that need to be synthesizedto optimize the final ribozyme equals the total number of positions tobe optimized divided by the number of positions (w) tested within eachClass. The number of pools in each Class=n^(w). The number ofClass=total number of positions/w.

[0046] In another preferred embodiment, the invention features a rapidmethod of screening for new catalytic nucleic acid motifs by keeping thebinding arms constant and varying one or more positions in a putativecatalytic domain. Applicant describes a method to vary positions withinthe catalytic domain, without changing positions within the bindingarms, in order to identify new catalytic motifs. An example isillustrated in FIG. 18. It is unclear how many positions are required toobtain a functional catalytic domain in a nucleic acid molecule, howeverit is reasonable to presume that if a large number of functionallydiverse nucleotide analogs can be used to construct the pools, arelatively small number of positions could constitute a functionalcatalytic domain. This may especially be true if analogs are chosen thatone would expect to participate in catalysis (e.g. acid/base catalysts,metal binding, etc.). In the example illustrated, four positions(designated 1, 2, 3 and 4) are chosen. In the first step, ribozymelibraries (Class 1) are constructed: position 1 is fixed (F₁) andpositions 2, 3 and 4 are random (X₂, X₃ and X₄, respectively). In step2, the pools (the number of pools tested depends on the number ofanalogs used; n) are assayed for activity. This testing may be performedin vitro or in a cellular or animal model. Whatever assay that is used,the pool with the desired characteristic is identified and libraries(class 2) are again synthesized with position 1 now constant (Z₁) withthe analog that was identified in class 1. In class 2, position 2 isfixed (F₂) and positions 3 and 4 are random (X₃ and X₄). This process isrepeated until every position has been made constant and the chemicalcomposition of the catalytic domain is determined. If the number ofpositions in the catalytic domain to be varied are large, then it ispossible to decrease the number of Classes by testing multiple positionswithin a single Class. the number of pools within a Class equals thenumber of nucleotides or analogs in the random mixture (ie. n) to the wpower, where w equals the number of positions fixed in each Class. Thenumber of Classes that need to be synthesized to optimize the finalribozyme equals the total number of positions to be optimized divided bythe number of positions (w) tested within each Class. The number ofpools in each Class=n^(w). The number of Classes=total number ofpositions/w.

[0047] In a preferred embodiment a method for identifying variants of anucleic acid catalyst is described comprising the steps of: a) selectingat least three (3) positions, preferably 3-12, specifically 4-10, withinsaid nucleic acid catalyst to be varied with a predetermined group ofdifferent nucleotides, these nucleotides are modified or unmodified(non-limiting examples of nucleotides that can used in this method areshown in FIG. 9); b) synthesizing a first class of different pools ofsaid nucleic acid catalyst, wherein the number of pools synthesized isequal to the number of nucleotides in the predetermined group ofdifferent nucleotides (for example if 10 different nucleotides areselected to be in the group of predetermined nucleotides then 10different pools of nucleic acid catalysts have to be synthesized),wherein at least one of the positions to be varied in each poolcomprises a defined nucleotide (fixed position; F) selected from thepredetermined group of different nucleotides and the remaining positionsto be varied comprise a random mixture of nucleotides (X positions)selected from the predetermined group of different nucleotides; c)testing the different pools of said nucleic acid catalyst underconditions suitable for said pools to show a desired attribute(including but not limited to improved cleavage rate, cellular andanimal efficacy, nuclease stability, enhanced delivery, desirablelocalization) and identifying the pool with said desired attribute andwherein the position with the defined nucleotide (F) in the pool withthe desired attribute is made constant (Z position) in subsequent steps;d) synthesizing a second class of different pools of nucleic acidcatalyst, wherein at least one of the positions to be varied in each ofthe second class of different pools comprises a defined nucleotide (F)selected from the predetermined group of different nucleotides and theremaining positions to be varied comprise a random mixture (X) ofnucleotides selected from the predetermined group of differentnucleotides (this second class of pools therefore has F, X and Zpositions); e) testing the second class of different pools of saidnucleic acid catalyst under conditions suitable for showing desiredattribute and identifying the pool with said desired attribute andwherein the position with the defined nucleotide in the pool with thedesired attribute is made constant (Z) in subsequent steps; and f) thisprocess is repeated until every position selected in said nucleic acidcatalyst to be varied is made constant.

[0048] In yet another preferred embodiment, a method for identifyingnovel nucleic acid molecules in a biological system is described,comprising the steps of: a) synthesizing a pool of nucleic acid catalystwith a substrate binding domain and a catalytic domain, wherein saidsubstrate binding domain comprises a random sequence; b) testing thepools of nucleic acid catalyst under conditions suitable for showing adesired effect (such as inhibition of cell proliferation, inhibition ofangiogenesis, modulation of growth and/or differentiation, and others)and identifying the catalyst with said desired attribute; c) using anoligonucleotide, comprising the sequence of the substrate binding domainof the nucleic acid catalyst showing said desired effect, as a probe,screening said biological system for nucleic acid moleculescomplementary to said probe; and d) isolating and sequencing saidcomplementary nucleic acid molecules. These nucleic acid moleculesidentified using a nucleic acid screening method described above may benew gene sequences, or known gene sequences. The advantage of thismethod is that nucleic acid sequences, such as genes, involved in abiological process, such as differentiation, cell growth, diseaseprocesses including cancer, tumor angiogenesis, arthritis,cardiovascular disease, inflammation, restenosis, vascular disease andthe like, can be readily identified.

[0049] In a preferred embodiment, the invention features a nucleic acidmolecule with catalytic activity having one of the formulae I-V:

[0050] Formula I

[0051] Formula II

[0052] Formula III

[0053] Formula IV

[0054] Formula V

[0055] In each of the above formulae, N represents independently anucleotide or a non-nucleotide linker, which may be same or different; Mand Q are independently oligonucleotides of length sufficient to stablyinteract (e.g., by forming hydrogen bonds with complementary nucleotidesin the target) with a target nucleic acid molecule (the target can be anRNA, DNA or RNA/DNA mixed polymers); preferably the length of Q isgreater than or equal to 3 nucleotides and the length of M is preferablygreater than or equal to 5 nucleotides; o and n are integers greaterthan or equal to 1 and preferably less than about 100, wherein if(N)_(o) and (N)_(n) are nucleotides, (N)o and (N)n are optionally ableto interact by hydrogen bond interaction; L is a linker which may bepresent or absent (i.e., the molecule is assembled from two separatemolecules), but when present, is a nucleotide and/or a non-nucleotidelinker, which may be a single-stranded and/or double-stranded region;and ______ represents a chemical linkage (e.g. a phosphate esterlinkage, amide linkage or others known in the art). 2′-O-MTM-U and2′-O-MTM-C refers to 2′-O-methylthiomethyl uridine and2′-O-methylthiomethyl-cytidine, respectively. A, C, U and G representadenosine, cytidine, uridine and guanosine nucleotides, respectively.The nucleotides in the formulae are unmodified or modified at the sugar,base, and/or phosphate portions as known in the art.

[0056] In yet another embodiment, the nucleotide linker (L) is a nucleicacid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tataptamer (TAR) and others (for a review see Gold et al., 1995, Annu. Rev.Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed.Gesteland and Atkins, pp 511, CSH Laboratory Press). A “nucleic acidaptamer” as used herein is meant to indicate nucleic acid sequencecapable of interacting with a ligand. The ligand can be any natural or asynthetic molecule, including but not limited to a resin, metabolites,nucleosides, nucleotides, drugs, toxins, transition state analogs,peptides, lipids, proteins, aminoacids, nucleic acid molecules,hormones, carbohydrates, receptors, cells, viruses, bacteria and others.

[0057] In yet another embodiment, the non-nucleotide linker (L) is asdefined herein.

[0058] The term “nucleotide” is used as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a sugar moiety.Nucleotide generally comprise a base, sugar and a phosphate group. Thenucleotides can be unmodified or modified at the sugar, phosphate and/orbase moiety, (also referred to interchangeably as nucleotide analogs,modified nucleotides, non-natural nucleotides, non-standard nucleotidesand other; see for example, Usman and McSwiggen, supra; Eckstein et al.,International PCT Publication No. WO 92/07065; Usman et al.,International PCT Publication No. WO 93/15187; all hereby incorporatedby reference herein). There are several examples of modified nucleicacid bases known in the art and has recently been summarized by Limbachet al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limitingexamples of base modifications that can be introduced into enzymaticnucleic acids without significantly effecting their catalytic activityinclude, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine) and others (Burgin et al., 1996, Biochemistry, 35,14090). By “modified bases” in this aspect is meant nucleotide basesother than adenine, guanine, cytosine and uracil at 1′ position or theirequivalents; such bases may be used within the catalytic core of theenzyme and/or in the substrate-binding regions.

[0059] In particular, the invention features modified ribozymes having abase substitution selected from pyridin4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouracil,naphthyl, 6-methyluracil and aminophenyl.

[0060] There are several examples in the art describing sugar andphosphate modifications that can be introduced into enzymatic nucleicacid molecules without significantly effecting catalysis and withsignificant enhancement in their nuclease stability and efficacy.Ribozymes are modified to enhance stability and/or enhance catalyticactivity by modification with nuclease resistant groups, for example,2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide basemodifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34;Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996Biochemistry 35, 14090). Sugar modification of enzymatic nucleic acidmolecules have been extensively described in the art (see Eckstein etal., International Publication PCT No. WO 92/07065; Perrault et al.Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317;Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman etal. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No.5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702).

[0061] Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into ribozymes without inhibiting catalysis,and are incorporated by reference herein. In view of such teachings,similar modifications can be used as described herein to modify thenucleic acid catalysts of the instant invention.

[0062] In yet another embodiment, the non-nucleotide linker (L) is asdefined herein. The term “non-nucleotide” as used herein include eitherabasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, or polyhydrocarbon compounds. Specific examplesinclude those described by Seela and Kaiser, Nucleic Acids Res. 1990,18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J.Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem.Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 andBiochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990,18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschkeet al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991,30:9914; Arnold et al., International Publication No. WO 89/02439; Usmanet al., International Publication No. WO 95/06731; Dudycz et al.,International Publication No. WO 95/11910 and Ferentz and Verdine, J.Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by referenceherein. Thus, in a preferred embodiment, the invention features anenzymatic nucleic acid molecule having one or more non-nucleotidemoieties, and having enzymatic activity to cleave an RNA or DNAmolecule. By the term “non-nucleotide” is meant any group or compoundwhich can be incorporated into a nucleic acid chain in the place of oneor more nucleotide units, including either sugar and/or phosphatesubstitutions, and allows the remaining bases to exhibit their enzymaticactivity. The group or compound is abasic in that it does not contain acommonly recognized nucleotide base, such as adenosine, guanine,cytosine, uracil or thymine. The terms “abasic” or “abasic nucleotide”as used herein encompass sugar moieties lacking a base or having otherchemical groups in place of base at the 1′ position.

[0063] In preferred embodiments, the enzymatic nucleic acid includes oneor more stretches of RNA, which provide the enzymatic activity of themolecule, linked to the non-nucleotide moiety. The necessary RNAcomponents are known in the art, see, e.g., Usman, supra. By RNA ismeant a molecule comprising at least one ribonucleotide residue.

[0064] As the term is used in this application,non-nucleotide-containing enzymatic nucleic acid means a nucleic acidmolecule that contains at least one non-nucleotide component whichreplaces a portion of a ribozyme, e.g., but not limited to, adouble-stranded stem, a single-stranded “catalytic core” sequence, asingle-stranded loop or a single-stranded recognition sequence. Thesemolecules are able to cleave (preferably, repeatedly cleave) separateRNA or DNA molecules in a nucleotide base sequence specific manner. Suchmolecules can also act to cleave intramolecularly if that is desired.Such enzymatic molecules can be targeted to virtually any RNAtranscript.

[0065] By the phrase “nucleic acid catalyst” is meant a nucleic acidmolecule capable of catalyzing (altering the velocity and/or rate of) avariety of reactions including the ability to repeatedly cleave otherseparate nucleic acid molecules (endonuclease activity) in a nucleotidebase sequence-specific manner. Such a molecule with endonucleaseactivity may have complementarity in a substrate binding region (e.g. Mand Q in formulae I-V) to a specified gene target, and also has anenzymatic activity that specifically cleaves RNA or DNA in that target.That is, the nucleic acid molecule with endonuclease activity is able tointramolecularly or intermolecularly cleave RNA or DNA and therebyinactivate a target RNA or DNA molecule. This complementarity functionsto allow sufficient hybridization of the enzymatic RNA molecule to thetarget RNA or DNA to allow the cleavage to occur. 100% complementarityis preferred, but complementarity as low as 50-75% may also be useful inthis invention. The nucleic acids may be modified at the base, sugar,and/or phosphate groups. The term enzymatic nucleic acid is usedinterchangeably with phrases such as ribozymes, catalytic RNA, enzymaticRNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNAenzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme orDNA enzyme. All of these terminologies describe nucleic acid moleculeswith enzymatic activity.

[0066] By “nucleic acid molecule” as used herein is meant a moleculecomprising nucleotides. The nucleic acid can be composed of modified orunmodified nucleotides or non-nucleotides or various mixtures andcombinations thereof.

[0067] By “complementarity” is meant a nucleic acid that can formhydrogen bond(s) with other RNA sequence by either traditionalWatson-Crick or other non-traditional types (for example, Hoogsteentype) of base-paired interactions.

[0068] By “oligonucleotide” as used herein, is meant a moleculecomprising two or more nucleotides.

[0069] The specific enzymatic nucleic acid molecules described in theinstant application are not limiting in the invention and those skilledin the art will recognize that all that is important in an enzymaticnucleic acid molecule of this invention is that it has a specificsubstrate binding site (e.g., M and/or Q of Formulae 1-V above) which iscomplementary to one or more of the target nucleic acid regions, andthat it have nucleotide sequences within or surrounding that substratebinding site which impart a nucleic acid cleaving activity to themolecule.

[0070] The invention provides a method for producing a class ofenzymatic cleaving agents which exhibit a high degree of specificity forthe nucleic acid sequence of a desired target. The enzymatic nucleicacid molecule is preferably targeted to a highly conserved sequenceregion of a target such that specific diagnosis and/or treatment of adisease or condition can be provided with a single enzymatic nucleicacid. Such enzymatic nucleic acid molecules can be delivered exogenouslyto specific cells as required. In the preferred hammerhead motif thesmall size (less than 60 nucleotides, preferably between 30-40nucleotides in length) of the molecule allows the cost of treatment tobe reduced.

[0071] Therapeutic ribozymes must remain stable within cells untiltranslation of the target RNA has been inhibited long enough to reducethe levels of the undesirable protein. This period of time variesbetween hours to days depending upon the disease state. Clearly,ribozymes must be resistant to nucleases in order to function aseffective intracellular therapeutic agents. Improvements in the chemicalsynthesis of RNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677;incorporated by reference herein) have expanded the ability to modifyribozymes to enhance their nuclease stability.

[0072] By “enzymatic portion” is meant that part of the ribozymeessential for cleavage of an RNA substrate.

[0073] By “substrate binding arm” is meant that portion of a ribozymewhich is complementary to (i.e., able to base-pair with) a portion ofits substrate. Generally, such complementarity is 100%, but can be lessif desired. For example, as few as 10 bases out of 14 may bebase-paired. Such arms are shown generally in FIG. 1A and as M and/or Qin Formulae I-V. That is, these arms contain sequences within a ribozymewhich are intended to bring ribozyme and target RNA together throughcomplementary base-pairing interactions.

[0074] In a preferred embodiment, the invention provides a method forproducing a class of enzymatic cleaving agents which exhibit a highdegree of specificity for the nucleic acid of a desired target. Suchenzymatic nucleic acid molecules can be delivered exogenously tospecific cells as required. Alternatively, the ribozymes can beexpressed from DNA/RNA vectors that are delivered to specific cells.

[0075] The enzymatic nucleic acid molecules of the instant invention canalso be expressed within cells from eukaryotic promoters (e.g., Izantand Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc.Natl. Acad. Sci. USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992 J. Virol, 66, 1432-41; Weerasinghe etal., 1991 J. Virol, 65, 5531-4; Ojwang et al., 1992 Proc. Natl. Acad.Sci. USA 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9;Sarver et al., 1990 Science 247, 1222-1225; Thompson et al., 1995Nucleic Acids Res. 23, 2259). Those skilled in the art realize that anynucleic acid can be expressed in eukaryotic cells from the appropriateDNA/RNA vector. The activity of such nucleic acids can be augmented bytheir release from the primary transcript by a ribozyme (Draper et al.,PCT WO 93/23569, and Sullivan et al., PCT WO94/02595; Ohkawa et al.,1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, NucleicAcids Res., 19, 5125-30; Ventura et al., 1993 Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994 J. Biol. Chem. 269, 25856; herebyincorporated in their totality by reference herein).

[0076] By “consists essentially of” is meant that the active ribozymecontains an enzymatic center or core equivalent to those in theexamples, and binding arms able to bind target nucleic acid moleculessuch that cleavage at the target site occurs. Other sequences may bepresent which do not interfere with such cleavage.

[0077] Thus, in one aspect, the invention features ribozymes thatinhibit gene expression and/or cell proliferation. These chemically orenzymatically synthesized nucleic acid molecules contain substratebinding domains that bind to accessible regions of specific targetnucleic acid molecules. The nucleic acid molecules also contain domainsthat catalyze the cleavage of target. Upon binding, the enzymaticnucleic acid molecules cleave the target molecules, preventing forexample, translation and protein accumulation. In the absence of theexpression of the target gene, cell proliferation, for example, isinhibited.

[0078] In a preferred embodiment, the enzymatic nucleic acid moleculesare added directly, or can be complexed with cationic lipids, packagedwithin liposomes, or otherwise delivered to smooth muscle cells. The RNAor RNA complexes can be locally administered to relevant tissues throughthe use of a catheter, infusion pump or stent, with or without theirincorporation in biopolymers. Using the methods described herein, otherenzymatic nucleic acid molecules that cleave target nucleic acid may bederived and used as described above. Specific examples of nucleic acidcatalysts of the instant invention are provided below in the Tables andfigures.

[0079] Sullivan, et al., supra, describes the general methods fordelivery of enzymatic RNA molecules. Ribozymes may be administered tocells by a variety of methods known to those familiar to the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such ashydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres. For some indications, ribozymes may be directly deliveredex vivo to cells or tissues with or without the aforementioned vehicles.Alternatively, the RNA/vehicle combination is locally delivered bydirect injection or by use of a catheter, infusion pump or stent. Otherroutes of delivery include, but are not limited to, intravascular,intramuscular, subcutaneous or joint injection, aerosol inhalation, oral(tablet or pill form), topical, systemic, ocular, intraperitoneal and/orintrathecal delivery. More detailed descriptions of ribozyme deliveryand administration are provided in Sullivan et al., supra and Draper etal., supra which have been incorporated by reference herein.

[0080] In another aspect of the invention, enzymatic nucleic acidmolecules that cleave target molecules are expressed from transcriptionunits inserted into DNA or RNA vectors. The recombinant vectors arepreferably DNA plasmids or viral vectors. Ribozyme expressing viralvectors could be constructed based on, but not limited to,adeno-associated virus, retrovirus, adenovirus, or alphavirus.Preferably, the recombinant vectors capable of expressing the ribozymesare delivered as described above, and persist in target cells.Alternatively, viral vectors may be used that provide for transientexpression of ribozymes. Such vectors might be repeatedly administeredas necessary. Once expressed, the ribozymes cleave the target mRNA.Delivery of ribozyme expressing vectors could be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from the patient followed by reintroduction into thepatient, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture and Stinchcomb, 1996,TIG., 12, 510).

[0081] In a preferred embodiment, an expression vector comprisingnucleic acid sequence encoding at least one of the nucleic acid catalystof the instant invention is disclosed. The nucleic acid sequenceencoding the nucleic acid catalyst of the instant invention is operablelinked in a manner which allows expression of that nucleic acidmolecule.

[0082] In one embodiment, the expression vector comprises: atranscription initiation region (e.g., eukaryotic pol I, II or IIIinitiation region); b) a transcription termination region (e.g.,eukaryotic pol I, II or III termination region); c) a gene encoding atleast one of the nucleic acid catalyst of the instant invention; andwherein said gene is operably linked to said initiation region and saidtermination region, in a manner which allows expression and/or deliveryof said nucleic acid molecule. The vector may optionally include an openreading frame (ORF) for a protein operably linked on the 5′ side or the3′-side of the gene encoding the nucleic acid catalyst of the invention;and/or an intron (intervening sequences).

[0083] By “patient” is meant an organism which is a donor or recipientof explanted cells or the cells themselves. “Patient” also refers to anorganism to which enzymatic nucleic acid molecules can be administered.Preferably, a patient is a mammal or mammalian cells. More preferably, apatient is a human or human cells.

[0084] By “vectors” is meant any nucleic acid- and/or viral-basedtechnique used to deliver a desired nucleic acid.

[0085] Another means of accumulating high concentrations of aribozyme(s) within cells is to incorporate the ribozyme-encodingsequences into a DNA or RNA expression vector. Transcription of theribozyme sequences are driven from a promoter for eukaryotic RNApolymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III(pol III). Transcripts from pol II or pol III promoters will beexpressed at high levels in all cells; the levels of a given pol IIpromoter in a given cell type will depend on the nature of the generegulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that ribozymesexpressed from such promoters can function in mammalian cells (e.g.Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al.,1992 Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992 NucleicAcids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. USA, 90,6340-4; L'Huillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al.,1993 Proc. Natl. Acad. Sci. U.S.A., 90, 8000-4; Thompson et al., 1995Nucleic Acids Res. 23, 2259; Sullenger & Cech, 1993, Science, 262,1566). The above ribozyme transcription units can be incorporated into avariety of vectors for introduction into mammalian cells, including butnot restricted to, plasmid DNA vectors, viral DNA vectors (such asadenovirus or adeno-associated virus vectors), or viral RNA vectors(such as retroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

[0086] In a preferred embodiment an expression vector comprising nucleicacid sequence encoding at least one of the catalytic nucleic acidmolecule of the invention, in a manner which allows expression of thatnucleic acid molecule.

[0087] The expression vector comprises in one embodiment; a) atranscription initiation region; b) a transcription termination region;c) a gene encoding at least one said nucleic acid molecule; and whereinsaid gene is operably linked to said initiation region and saidtermination region, in a manner which allows expression and/or deliveryof said nucleic acid molecule. In another preferred embodiment theexpression vector comprises: a) a transcription initiation region; b) atranscription termination region; c) an open reading frame; d) a geneencoding at least one said nucleic acid molecule, wherein said gene isoperably linked to the 3′-end of said open reading frame; and whereinsaid gene is operably linked to said initiation region, said openreading frame and said termination region, in a manner which allowsexpression and/or delivery of said nucleic acid molecule. In yet anotherembodiment the expression vector comprises: a) a transcriptioninitiation region; b) a transcription termination region; c) an intron;d) a gene encoding at least one said nucleic acid molecule; and whereinsaid gene is operably linked to said initiation region, said intron andsaid termination region, in a manner which allows expression and/ordelivery of said nucleic acid molecule. In other embodiment, theexpression vector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; d) an open readingframe; e) a gene encoding at least one said nucleic acid molecule,wherein said gene is operably linked to the 3′-end of said open readingframe; and wherein said gene is operably linked to said initiationregion, said intron, said open reading frame and said terminationregion, in a manner which allows expression and/or delivery of saidnucleic acid molecule.

[0088] In a preferred embodiment, the invention features a method ofsynthesis of enzymatic nucleic acid molecules of instant invention whichfollows the procedure for normal chemical synthesis of RNA as describedin Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al.,1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 NucleicAcids Res. 23, 2677-2684 and makes use of common nucleic acid protectingand coupling groups, such as dimethoxytrityl at the 5′-end, andphosphoramidites at the 3′-end. Small scale synthesis were conducted ona 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 μmolscale protocol with a 5 min coupling step for alkylsilyl protectednucleotides and 2.5 min coupling step for 2′-O-methylated nucleotides.Table II outlines the amounts, and the contact times, of the reagentsused in the synthesis cycle. A 6.5-fold excess (163 μL of 0.1 M=16.3μmol) of phosphoramidite and a 24-fold excess of S-ethyl tetrazole (238μL of 0.25 M=59.5 μmol) relative to polymer-bound 5′-hydroxyl is used ineach coupling cycle. Average coupling yields on the 394 AppliedBiosystems, Inc. synthesizer, determined by colorimetric quantitation ofthe trityl fractions, is 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer: detritylationsolution was 2% TCA in methylene chloride (ABI); capping was performedwith 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10%2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM I₂, 49 mMpyridine, 9% water in THF (Millipore). B & J Synthesis Gradeacetonitrile is used directly from the reagent bottle. S-Ethyl tetrazolesolution (0.25 M in acetonitrile) is made up from the solid obtainedfrom American International Chemical, Inc.

[0089] In a preferred embodiment, deprotection of the chemicallysynthesized nucleic acid catalysts of the invention is performed asfollows. The polymer-bound oligoribonucleotide, trityl-off, istransferred from the synthesis column to a 4 mL glass screw top vial andsuspended in a solution of methylamine (MA) at 65° C. for 10 min. Aftercooling to −20° C., the supernatant is removed from the polymer support.The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1,vortexed and the supernatant is then added to the first supernatant. Thecombined supernatants, containing the oligoribonucleotide, are dried toa white powder.

[0090] The base-deprotected oligoribonucleotide is resuspended inanhydrous TEA·HF/NMP solution (250 μL of a solution of 1.5 mLN-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA·3 HF to provide a 1.4MHF concentration) and heated to 65° C. for 1.5 h. The resulting, fullydeprotected, oligomer is quenched with 50 mM TEAB (9 mL) prior to anionexchange desalting.

[0091] For anion exchange desalting of the deprotected oligomer, theTEAB solution is loaded on to a Qiagen 500® anion exchange cartridge(Qiagen Inc.) that is pre-washed with 50 mM TEAB (10 mL). After washingthe loaded cartridge with 50 mM TEAB (10 mL), the RNA is eluted with 2 MTEAB (10 mL) and dried down to a white powder. The average stepwisecoupling yields are generally >98% (Wincott et al., 1995 Nucleic AcidsRes. 23, 2677-2684).

[0092] Ribozymes of the instant invention are also synthesized from DNAtemplates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck,1989, Methods Enzymol. 180, 51).

[0093] Ribozymes are purified by gel electrophoresis using generalmethods or are purified by high pressure liquid chromatography (HPLC;See Wincott et al., supra) the totality of which is hereby incorporatedherein by reference) and are resuspended in water.

[0094] In another preferred embodiment, catalytic activity of themolecules described in the instant invention can be optimized asdescribed by Draper et al., supra. The details will not be repeatedhere, but include altering the length of the ribozyme binding arms, orchemically synthesizing ribozymes with modifications (base, sugar and/orphosphate) that prevent their degradation by serum ribonucleases and/orenhance their enzymatic activity (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al., 1990 Nature344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren,1992 Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; and Rossi et al., International PublicationNo. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al.,supra; all of these describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of enzymatic RNAmolecules). Modifications which enhance their efficacy in cells, andremoval of bases from stem loop structures to shorten RNA synthesistimes and reduce chemical requirements are desired. (All thesepublications are hereby incorporated by reference herein.).

[0095] By “enhanced enzymatic activity” is meant to include activitymeasured in cells and/or in vivo where the activity is a reflection ofboth catalytic activity and ribozyme stability. In this invention, theproduct of these properties is increased or not significantly (less that10 fold) decreased in vivo compared to an all RNA ribozyme.

[0096] In yet another preferred embodiment, nucleic acid catalystshaving chemical modifications which maintain or enhance enzymaticactivity is provided. Such nucleic acid is also generally more resistantto nucleases than unmodified nucleic acid. Thus, in a cell and/or invivo the activity may not be significantly lowered. As exemplifiedherein such ribozymes are useful in a cell and/or in vivo even ifactivity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry,35, 14090). Such ribozymes herein are said to “maintain” the enzymaticactivity on all RNA ribozyme.

[0097] In a most preferred embodiment the invention features a method ofsynthesizing ribozyme libraries of various sizes. This inventiondescribes methods to chemically synthesize ribozyme libraries of varioussizes from suitable nucleoside analogs.

[0098] Considerations for the selection of nucleotide building blocksand determination of coupling efficiency: In addition to structuralconsiderations (hydrogen bond donors and acceptors, stacking properties,pucker orientation of sugars, hydrophobicity or hydrophilicity of somesubgroups constitutive of the nucleotides) that may lead to theselection of a specific nucleotide to be included in the design of aribozyme library, one of the important features that needs to beconsidered when selecting nucleotide building blocks is the chemicalcompatibility of such building blocks with ribozyme synthesis. A“nucleotide building block” is a nucleoside or nucleoside analog thatpossess a suitably protected phosphorus atom at the oxidation state Vreacting readily, upon activation, to give a P^(V)-containinginternucleoside linkage. A suitable nucleoside building block may alsocontain a phosphorus atom at the oxidation state III reacting readily,upon activation, to give a P^(III)-containing internucleoside linkagethat can be oxidized to the desired P^(V)-containing internucleosidelinkage. Applicant has found that the phosphoramidite chemistry(P^(III)) is a preferred coupling method for ribozyme library synthesis.There are several other considerations while designing and synthesizingcertain ribozyme libraries, such as: a) the coupling efficiencies of thenucleotide building blocks considered for a ribozyme library should notfall below 90% to provide a majority of full-length ribozyme; b) thenucleotide building blocks should be chemically stable to the selectedsynthesis and deprotection conditions of the particular ribozymelibrary; c) the deprotection schemes for the nucleotide building blocksincorporated into a ribozyme library, should be relatively similar andbe fully compatible with ribozyme deprotection protocols. In particular,nucleoside building blocks requiring extended deprotection or thatcannot sustain harsh treatment should be avoided in the synthesis of aribozyme library. Typically, the reactivity of the nucleotide buildingblocks should be optimum when diluted to 100 mM to 200 mM in non-proticand relatively polar solvent. Also the deprotection condition using 3:1mixture of ethanol and concentrated aqueous ammonia at 65 degrees C. for4 hours followed by a fluoride treatment as exemplified in Wincott etal. supra, is particularly useful for ribozyme synthesis and is apreferred deprotection pathway for such nucleotide building blocks.

[0099] In one preferred embodiment, a “nucleotide building block mixing”approach to generate ribozyme libraries is described. This methodinvolves mixing various nucleotide building blocks together inproportions necessary to ensure equal representation of each of thenucleotide building blocks in the mixture. This mixture is incorporatedinto the ribozyme at position(s) selected for randomization.

[0100] The nucleotide building blocks selected for incorporation into aribozyme library, are typically mixed together in appropriateconcentrations, in reagents, such as anhydrous acetonitrile, to form amixture with a desired phosphoramidite concentration. This approach forcombinatorial synthesis of a ribozyme library with one or more randompositions within the ribozyme (X as described above) is particularlyuseful since a standard DNA synthesizer can handle a building blockmixture similar to a building block solution containing a singlebuilding block. Such a nucleotide building block mixture is coupled to asolid support or to a growing ribozyme sequence attached to asolid-support. To ensure that the ribozyme library synthesized achievesthe desired complexity, the scale of the synthesis is increasedsubstantially above that of the total complexity of the library. Forexample, a 2.5 μmole ribozyme synthesis provides ˜3×10¹⁷ ribozymemolecules corresponding to sub-nanomolar amounts of each member of abillion compounds ribozyme library.

[0101] Divinylbenzene highly cross-linked polystyrene solid-supportconstitutes the preferred stationary phase for ribozyme librarysynthesis. However, other solid-support systems utilized in DNA or RNAsynthesis can also be used for ribozyme library synthesis. This includessilica-based solid-supports such as controlled-pore glass (CPG) orpolymeric solid-supports such as all types of derivatized polystyreneresins, grafted polymers of chloromethylated polystyrene crosslinkedwith ethylene glycol, oligoethylene glycol.

[0102] Because of different coupling kinetics of the nucleotide buildingblocks present in a mixture, it is necessary to evaluate the relativeincorporation of each of the members of the mixture and to adjust, ifneeded, the relative concentration of the building blocks in the mixtureto get equimolar representation, compensating thereby the kineticparameter. Typically a building block that presents a slow couplingkinetic will be over-represented in the mixture and vice versa for abuilding block that presents a fast coupling kinetic. When equimolarincorporation is sought, acceptable limits for unequal incorporation maygenerally be +/−10%.

[0103] Synthesis of a random ribozyme library can be performed eitherwith the mixture of desired nucleotide building blocks (phosphoramiditepooling protocol), or with a combination of certain random positions(obtained by using one or more building block mixtures) and one or morefixed positions that can be introduced through the incorporation of asingle nucleotide building block reagent. For instance, in theoligonucleotide model 5′-TT XXXX TTB-3′ used in example 2 infra, thepositions from 3′-end 1 is fixed as 2′-deoxy-inverted abasic ribose (B),positions 2, 3, 8 and 9 have been fixed as 2′-deoxy-thymidine (T) whilethe X positions 4-7 correspond to an approximately equimolardistribution of all the nucleotide building blocks that make up the Xmixture.

[0104] In another preferred embodiment, a “mix and split” approach togenerate ribozyme libraries is described. This method is particularlyuseful when the number of selected nucleotide building blocks to beincluded in the library is large and diverse (greater than 5 nucleotidebuilding blocks) and/or when the coupling kinetics of the selectednucleotide building blocks do not allow competitive coupling even afterrelative concentration adjustments and optimization. This methodinvolves a multi-step process wherein the solid support used forribozyme library synthesis is “split” (divided) into equal portions,(the number of portions is equal to the number of different nucleotidebuilding blocks (n) chosen for incorporation at one or more randompositions within the ribozyme). For example, if there are 10 differentnucleotide building blocks chosen for incorporation at one or morepositions in the ribozyme library, then the solid support is dividedinto 10 different portions. Each portion is independently coupled to oneof the selected nucleotide building blocks followed by mixing of all theportions of solid support. The ribozyme synthesis is then resumed asbefore the division of the building blocks. This enables the synthesisof a ribozyme library wherein one or more positions within the ribozymeis random. The number of “splitting” and “mixing” steps is dependent onthe number of positions that are random within the ribozyme. For exampleif three positions are desired to be random then three differentsplitting and mixing steps are necessary to synthesize the ribozymelibrary.

[0105] Random ribozyme libraries are synthesized using a non-competitivecoupling procedure where each of the selected nucleotide analogs “n”separately couple to an inverse “n” (1/n) number of aliquots ofsolid-support or of a growing ribozyme chain on the solid-support. Avery convenient way to verify completeness of the coupling reaction isthe use of a standard spectrophotometric DMT assay (OligonucleotideSynthesis, A Practical Approach, ed. M. Gait, pp 48, IRC Press, Oxford,UK; incorporated by reference herein). These aliquots may besubsequently combined, mixed and split into one new aliquot. A similarapproach to making oligonucleotide libraries has recently been describedby Cook et al., (U.S. Pat. No. 5,587,471) and is incorporated byreference herein.

EXAMPLES

[0106] The following are non-limiting examples showing the synthesis,screening and testing of catalytic nucleic acids of the instantinvention.

[0107] The development of nucleic acid catalysts that are optimal forcatalytic activity would contribute significantly to any strategy thatemploys nucleic acid cleaving ribozymes for the purpose of regulatinggene expression. The hammerhead ribozyme functions with a catalytic rate(k_(cat)) of ˜1 min⁻¹ in the presence of saturating (10 mM)concentrations of Mg²⁺ cofactor. However, the rate for this ribozyme inMg²⁺ concentrations that are closer to those found inside cells (0.5-2mM) may be 10- to 100-fold slower. In contrast, the RNase P holoenzymeis believed to catalyze pre-tRNA cleavage with a k_(cat) of ˜30 min⁻¹under optimal assay conditions. An artificial ‘RNA ligase’ ribozyme hasbeen shown to catalyze the corresponding self-modification reaction witha rate of ˜100 min⁻¹ (Ekland et al., 1995, Science, 269, 364). Finally,replacement of a specific residue within the catalytic core of thehammerhead with certain nucleotide analogues gives modified ribozymesthat show as much as a 10-fold improvement in catalytic rate (Burgin etal., 1996, supra). These findings demonstrate that ribozymes can promotechemical transformations with catalytic rates that are significantlygreater than those displayed in vitro by most natural self-cleavingribozymes. It is then possible that the structures of certain ribozymesmay not be optimized to give maximal catalytic activity, or thatentirely new nucleic acid catalysts could be made that displaysignificantly faster rates of catalysis.

[0108] An extensive array of site-directed mutagenesis studies have beenconducted with ribozymes, such as the hammerhead ribozyme, to proberelationships between nucleotide sequence and catalytic activity. Thesesystematic studies have made clear that most nucleotides in theconserved core of a ribozyme (Forster & Symons, 1987, Cell, 49, 211;Ruffner et al., 1990, Biochemistry 29, 10695; Couture et al., 1990, J.Mol. Bio. 215, 345; Berzal-Herranz et al., 1993 supra; Perrota et al.,1996, Nucleic Acid Res. 24,1314) cannot be mutated without significantloss of catalytic activity. In contrast, a selection strategy thatsimultaneously surveys a large pool of mutagenized ribozymes for theones that retain catalytic activity could be used more efficiently todefine immutable sequences and to identify new ribozyme variants(Breaker, 1997, supra). For example, Joseph and Burke (1993; J. Biol.Chem., 268, 24515) have used an in vitro selection approach to rapidlysurvey for sequence variants of the ‘hairpin’ self-cleaving RNA thatshow improved catalytic activity. This approach was successful inidentifying two mutations in the hairpin ribozyme that together give a10-fold improvement in catalytic rate. Although similar in vitroselection experiments have been conducted with the hammerhead ribozyme(Nakamaye & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra;Ishizaka et al., 1995, supra), none of these efforts have successfullyscreened full-sized hammerhead ribozymes for all possible combinationsof sequence variants that encompass the entire catalytic core.

Example 1 Optimizing Loop II Sequence of a Hammerhead Ribozyme (HH-B)for Enhanced Catalytic Rates

[0109] To test the feasibility of the combinatorial approach describedin FIG. 6 approach, Applicant chose to optimize the sequence of loop-IIof a hammerhead ribozyme (HH-B) (see FIG. 13). Previous studies haddemonstrated that a variety of chemical modifications and differentsequences within loop-II may have significant effects on the rate ofcleavage in vitro, despite the fact that this sequence is notphylogenetically conserved and can in fact be deleted completely.According to the standard numbering system for the hammerhead ribozyme,the four positions within loop II are numbered I2.1, I2.2, I2.3, andI2.4. The Starting Ribozyme (HH-B) contained the sequenceG_(I2.1)A_(I2.2)A_(I2.3)A_(I2.4). For simplicity, the four positionswill be numbered 5′ to 3′: G_(I2.1)=1; A_(I2.2)=2; A_(I2.3)=3;A_(I2.4)=4. The remainder of the hammerhead ribozyme “template” remainedconstant and is based on a previously described hammerhead motif (Draperet al., International PCT Publication No. WO 95/13380, incorporated byreference herein).

[0110] A strategy for optimizing the four (number of Classes=4) loop-IIpositions is illustrated in FIG. 14. The four standard ribosenucleotides (A, C, U and G) were chosen to construct the ribozyme pools(n=4). In the first step, four different pools were synthesized by thenucleotide building block mixing approach described herein. Applicantfirst chose to “fix” (designated F) position 3 because preliminaryexperiments indicated that the identity of the base at this position hadthe most profound effects on activity; positions 1, 2 and 4 are random.The four pools were assayed under stoichiometric conditions (1 μMribozyme; 1 μM substrate), to help ensure that the entire population ofribozymes in each pool was assayed. Substrate and ribozyme werepre-annealed and the reactions were initiated with the addition of 10 mMMgCl₂. The rate of cleavage for each library was derived from plots offraction of substrate cleaved as a function of time. Reactions were alsoperformed simultaneously with the starting ribozyme (i.e. homogenous,loop-II=GAAA). The relative rate of cleavage for each library (k_(rel))was calculated by dividing the observed rate of the library by the rateof the control/starting ribozyme and is plotted in FIG. 15. The errorbars indicate the standard error derived from the curve fits. Theresults show that all four pools had similar rates (k_(rel)); however,the library possessing “U” at position 3 was slightly faster.

[0111] Ribozyme pools were again synthesized (Class 2) with position 3being made constant (U₃), position 4 was fixed (F₄) and positions 1 and2 were random (X). The four pools were assayed as before; the poolcontaining “A” at position 4 was identified as the most desirable pool.Therefore, during the synthesis of the next pool (Class 3), positions 3and 4 were constant with U₃ and A₄, position 2 was fixed (F₂) andposition 1 was random (X). The four pools were again assayed; all fourpools showed very similar, but substantially elevated rates of cleavage.The pool containing U at position 2 was identified as the fastest.Therefore, during the synthesis of the final four ribozymes (Class 4),position 3, 4 and 2 were made constant with U₃, A₄ and U₂; position 1was fixed with A, U, C or G. The final ribozyme containing G at position4 was clearly identified as the fastest ribozyme, allowing theidentification of G_(I2.1) U_(I2.2) U_(I2.3) A_(I2.4) as the optimizedribozyme motif.

[0112] To confirm that the final ribozyme (G_(I2.1) U_(I2.2) U_(I2.3)A_(I2.4)) was indeed faster that the starting ribozyme (G_(I2.1)A_(I2.2) A_(I2.3) A_(I2.4)), we compared the two ribozymes (illustratedin FIG. 16) under single-turnover conditions at saturating ribozymeconcentrations. The observed rates should therefore measure the rate ofthe chemical step, k₂. The fraction of substrate remaining uncleaved asa function of time is shown in FIG. 16 (lower panel), and the derivedrate contents are shown. The results show that the optimized ribozymecleaves >10 times faster (3.7 min⁻¹ vs. 0.35 min⁻¹) than the startingribozyme.

Example 2 Optimizing Core Chemistry of a Hammerhead Ribozyme (HH-A)

[0113] To further test the feasibility of the approach described in FIG.6, we chose to optimize the three pyrimidine residues within the core ofa hammerhead ribozyme (HH-A). These three positions (shown in FIG. 7 asU7, U4 and C3) were chosen because previous studies indicated that thesepositions are critical for both stability (Beigelman et al., 1995,supra) and activity (Ruffner et al., 1990, supra; Burgin et al., 1996,supra) of the ribozyme. According to the standard numbering system forthe hammerhead ribozyme, the three pyrimidine positions are 7, 4 and 3.For construction of the libraries, the ribozyme positions are numbered3′ to 5′: position 24=7, position 27=4, and position 28=3 (see FIG. 7).The remainder of the hammerhead ribozyme “template” remained constantand is based on a previously described hammerhead motif (Thompson etal., U.S. Pat. No. 5,610,052, incorporated by reference herein). Thestarting ribozyme template is targeted against nucleotide position 823of k-ras mRNA (Site A). Down regulation of this message, as a result ofribozyme action, results in the inability of the cells to proliferate.Therefore in order to optimize a ribozyme, we chose to identify“variants” which were successful in inhibiting cell proliferation.

[0114] Cell Culture Assay:

[0115] Ribozyme:lipid Complex Formation

[0116] Ribozymes and LipofectAMINE were combined DMEM at finalconcentrations of 100 nM and 3.6 μM, respectively. Complexes wereallowed to form for 15 min at 37 C. in the absence of serum andantibiotics.

[0117] Proliferation Assay

[0118] Primary rat aortic smooth muscle cells (RASMC) were seeded at adensity of 2500 cells/well in 48 well plates. Cells were incubatedovernight in DMEM, supplemented with 20% fetal bovine serum (FBS),Na-pyruvate, penicillin (50 U/ml), and streptomycin (50 μg/ml).Subsequently cells were rendered quiescent by a 48 h incubation in DMEMwith 0.5% FBS.

[0119] Cells were incubated for 1.5 h with serum-free DMEMribozyme:lipid complexes. The medium was replaced and cells wereincubated for 24 h in DMEM with 0.25% FCS.

[0120] Cells were then stimulated with 10% FBS for 24 h. ³H-thymidine(0.3 μCi//well) was present for the last 12 h of serum stimulation.

[0121] At the end of the stimulation period the medium was aspirated andcells were fixed in icecold TCA (10%) for 15 min. The TCA solution wasremoved and wells were washed once with water. DNA was extracted byincubation with 0.1 N NaOH at RT for 15 min. Solubilized DNA wasquantitatively transferred to minivials. Plates were washed once withwater. Finally, ³H-thymidine incorporation was determined by liquidscintillation counting.

[0122] A strategy for optimizing the three (number of Class=3)pyrimidine residues is illustrated in FIG. 8. Ten different nucleotideanalogs (illustrated in FIG. 9) were chosen to construct the ribozymelibrary (n=10). In the first step, ten different pools (Class 1) weresynthesized by the mix and split approach described herein. Positions 24and 27 were random and position 28 was fixed with each of the tendifferent analogs. The ten different pools were formulated with acationic lipid (Jarvis et al., 1996, RNA, 2,419; incorporated byreference herein), delivered to cells in vitro, and cell proliferationwas subsequently assayed (see FIG. 10). A positive control (activeribozyme) inhibited cell proliferation by ˜50% and an inactive control(inactive) resulted in a less than 25% reduction in cell proliferation.The ten ribozyme pools resulted in intermediate levels of reduction.However, the best pool could be identified as X₂₄ X₂₇ 2′-MTM-U₂₈(positions 24 and 27 random; 2′-O-MTM-U at position 28). Therefore, asecond ribozyme library (Class 2) was synthesized with position 28constant (2′-O-MTM-U); position 24 was random (X₂₄) and position 27 wasfixed with each of the ten different analogs (F₂₇). Again, the ten poolswere assayed for their ability to inhibit cell proliferation. AmongClass 2, two pools inhibited proliferation equally well: X₂₄2′-C-allyl-U₂₇ 2′-O-MTM-U₂₈ and X₂₄ 2′-O-MTM-C₂₇ 2′-O-MTM-U₂₈. Because asingle “winner” could not be identified in Class 2, position 27 was madeconstant with either 2′-C-allyl-U or with 2′-O-MTM-C and the ten analogswere placed individually at position 24 (Class 3). Therefore in Class 3,twenty different ribozymes were assayed for their ability to inhibitcell proliferation. Because both positions 27 and 28 are constant, thefinal twenty ribozymes contain no random positions. Thus in the finalgroup (Class 3), pure ribozymes and not pools were assayed. Among thefinal groups four ribozymes inhibited cell proliferation to a greaterextent than the control ribozyme (FIG. 10). These four winners areillustrated in FIG. 11A. FIG. 11B shows general formula for fourdifferent motifs. A formula for a novel ribozyme motif is shown in FIG.12.

Example 3 Identifying Accessible Sites for Ribozyme Action in a Target

[0123] In the previous two examples (1 and 2), positions within thecatalytic domain of the hammerhead ribozyme were optimized. The numberof groups that needed to be tested equals=the total number of positionswithin the ribozyme that were chosen to be tested. A similar procedurecan be used on the binding arms of the ribozyme. The sequence of thebinding arms determines the site of action of the ribozyme. Thecombinatorial approach can be used to identify those sites byessentially testing all possible arm sequences. The difficulty with thisapproach is that ribozymes require a certain number of base pairs(12-16) in order bind tightly and specifically. According to theprocedure outlined above, this would require 12-16 different groups ofribozyme pools; 12-16 positions would have to be optimized which wouldrequire 12-16 different groups being synthesized and tested. Each poolwould contain the four different nucleotides (A, C, U and G) ornucleotide analogs (n=4). It would be very time consuming to test eachgroup, identify the best pool, synthesize another group of ribozymepools with one additional position constant, and then repeat theprocedure until all 12-16 groups had been tested. However it is possibleto decrease the number of groups by testing multiple positions within asingle group. In this case, the number of pools within a group equalsthe number of nucleotides or analogs in the random mixture (i.e. n) tothe w power, where w equals the number of positions fixed in each group.The number of groups that need to be synthesized to optimize the finalribozyme equals the total number of positions to be optimized divided bythe number of positions (w) tested within each group. The number ofpools in each group=n^(w). The number of groups=total number ofpositions/w.

[0124] For example, FIG. 17 illustrates this concept on a hammerheadribozyme containing 12 base pair binding arms. Each of the two bindingarms form 6 base pairs with it's corresponding RNA target. It isimportant to note that for the hammerhead ribozyme one residue (A15.1)must remain constant; A15.1 forms a base pair with a substratenucleotide (U16.1) but is also absolutely required for ribozymeactivity. It is the only residue within the hammerhead ribozyme that ispart of both the catalytic domain, and the binding domain (arms). In theexample this position is not optimized. In the first Group, threepositions are fixed (designated F) with the four different 2′-O-methylnucleotides (A, C, U and G). The 2′-O-methyl modification stabilizes theribozyme against nuclease degradation and increases the binding affinitywith it's substrate. The total number of pools in each group does notequal n, as in the previous examples. The number of pools in each groupequals 4³=(four analogs)^(Λ)(number of positions fixed; 3)=64. In all 64pools, all other positions in the arm are made random (designated X) bythe nucleotide mixing building block approach. The catalytic domain isnot considered in this example and therefore remains part of theribozyme template (i.e. constant).

[0125] In the first step, all 64 ribozyme pools are tested. This testmay be cleavage in vitro (see Example 1), or efficacy in a cellular (seeExample 2) or animal model, or any other assayable end-point. Thisend-point however, should be specific to a particular RNA target. Forexample, if one wishes to identify accessible sites within the mRNA ofGeneB, a suitable end-point would be to look for decreased levels ofGeneB mRNA after ribozyme treatment. After a winning pool is identified,since each pool specifies the identity of three positions (w), threepositions can be made constant for the next group (Class 2). Class 2 issynthesized containing 64 different pools; three positions that werefixed in Class 1 are now constant (designated Z), three more positionsare fixed (F), and the remaining positions (X) are a random mix of thefour nucleotides. The 64 pools are assayed as before, a winning pool isidentified, allowing three more positions to be constant in the nextClass of ribozyme pools (Class 3) and the process is repeated again. Inthe final Class of ribozymes (Class 4), only two positions are fixed,all other positions have been previously fixed. The total number ofribozymes is therefore n^(w)=4²=16; these ribozymes also contain norandom positions. In the final step (step 4), the 16 ribozymes aretested; the winning ribozyme defines the sequence of the binding armsfor a particular target.

[0126] Fixing multiple positions within a single group it is possible todecrease the overall number of groups that need to be tested. Asmentioned, this is particularly useful when a large number of differentpositions need to be optimized. A second advantage to this approach isthat it decreases the complexity of molecules in each pool. If one wouldexpect that many combinations within a given pool will be inactive, bydecreasing the number of different ribozymes in each pool, it will beeasier to identify the “winning” pool. In this approach, a larger numberof pools have to be tested in each group, however, the number of groupsis smaller and the complexity of each ribozyme pool is smaller. Finally,it should be emphasized there is not a restriction on the number ofpositions or analogs that can be tested. There is also no restriction onhow many positions are tested in each group.

Example 4 Identifying New RNA Targets for Ribozymes

[0127] As described above for identifying ribozyme-accessible sites, theassayed used to identify the “winning” pool of ribozymes is not definedand may be cleavage in vitro (see Example 1), or efficacy in a cellular(see Example 2) or animal model, or any other assayable end-point. Foridentifying accessible sites, this end-point should be specific to aparticular RNA target (e.g. mRNA levels). However, the end-point couldalso be nonspecific. For example, one could choose a disease model andsimply identify the winning ribozyme pool based on the ability toprovide a desired effect. In this case, it is not even necessary to knowwhat the cellular target that is being acted upon by the ribozyme is.One can simply identify a ribozyme that has a desired effect. Theadvantage to this approach is that the sequence of the binding arms willbe complementary to the RNA target. It is therefore possible to identifygene products that are involved in a disease process or any otherassayable phenotype. One does not have to know what the target is priorto starting the study. The process of identifying an optimized ribozyme(arm combinatorial) identifies both the drug (ribozyme) and the RNAtarget, which may be a known RNA sequence or a novel sequence leading tothe discovery of new genes.

Example 5 Identifying New Ribozyme Catalytic Domains

[0128] In the previous two examples, positions within the binding domainof the hammerhead ribozyme were varied and positions within thecatalytic domain were not changed. Conversely, it is possible to varypositions within the catalytic domain, without changing positions withinthe binding arms, in order to identify new catalytic motifs. An exampleis illustrated in FIG. 18. The hammerhead ribozyme, for examplecomprises about 23 residues within the catalytic domain. It is unclearhow many of these 23 positions are required to obtain a functionalcatalytic domain, however it is reasonable to presume that if a largenumber of functionally diverse nucleotide analogs can be used toconstruct the pools, a relatively small number of positions couldconstitute a functional catalytic domain. This may especially be true ifanalogs are chosen that one would expect to participate in catalysis(e.g. acid/base catalysts, metal binding, etc.). In the exampleillustrated in FIG. 18, four positions (designated 1, 2, 3 and 4) arechosen. In the first step, ribozyme libraries (Class 1) are constructed:position 1 is fixed (F₁) and positions 2, 3 and 4 are random (X₂, X₃ andX₄, respectively). In step 2, the pools (the number of pools testeddepends on the number of analogs used; n) are assayed for activity. Thistesting may be performed in vitro or in a cellular or animal model.Whatever assay that is used, the pool with the most activity isidentified and libraries (class 2) are again synthesized with position 1now constant (Z₁) with the analog that was identified in class 1. Inclass 2, position 2 is fixed (F₂) and positions 3 and 4 are random (X₃and X₄). This process is repeated until every position has been madeconstant, thus identifying the catalytic domain or a new motif.

Example 6 Determination of Coupling Efficiency of the PhosphoramiditeDerivatives of 2′-C-allyl-uridine, 1; 4′-thio-cytidine, 2;2′-methylthiomethyl-uridine, 3; 2′-methylthiomethyl-cytidine, 4;2′-amino-uridine, 5; N3-methyl-uridine, 6;1-β-D-(ribofuranosyl)-pyridin4-one, 7;1-β-D-(ribofuranosyl)-pyridin-2-one, 8; 1-β-D-(ribofuranosyl)-phenyl, 9;6-methyl-uridine, 10 to be used in a Split and Mix Approach

[0129] The determination of the coupling efficiency of amidites 1 to 10was assessed using ten model sequences agacXGAuGa (where upper caserepresents ribonucleotide residues, lower case represents 2′-O-methylribonucleotide residues and X is amidites 1 to 10, to be used in theconstruction of a hammerhead ribozyme library wherein the modifiedamidites 1 to 10 would be incorporated. Ten model sequences weresynthesized using ten 0.112 g aliquots of 5′-O-DMT-2′-O-Me-AdenosinePolystyrene (PS) solid-support loaded at 22.3 μmol/g and equivalent to a2.5 μmol scale synthesis. Synthesis of these ten decamers were performedon ABI 394 DNA synthesizer (Applied Biosystems, Foster City, Calif.)using standard nucleic acid synthesis reagents and synthesis protocols,with the exception of an extended (7.5 min) coupling time for theribonucleoside phosphoramidites and phosphoramidites 1, 2, 3, 4, 6, 7,8, 9, 10, 12.5 min coupling time for the 2′-amino-uridinephosphoramidite, amidite 5 and 2.5 min coupling time for the 2′-O-methylnucleoside phosphoramidites.

[0130] Oligomers were cleaved from the solid support by treatment with a3:1 mixture of ammonium hydroxide:absolute ethanol at 65 degree C. for 4hrs followed by a desilylation treatment and butanol precipitation asdescribed in Wincott et al. (Wincott et al, Nucleic Acids Res, 1995, 23,2677-2684; incorporated by reference herein). Oligonucleotides wereanalyzed directly on an anion-exchange HPLC column (Dionex, Nucleopac,PA-100, 4×250 mm) using a gradient of 50% to 80% of B over 12 minutes(A=10 mM sodium perchlorate, 1 mM Tris, pH 9.43; B=300 mM sodiumperchlorate, 1 mM Tris, pH 9.36) and a Hewlett-Packard 1090 HPLC system.

[0131] The average stepwise yield (ASWY), indicating the couplingefficiency of phosphoramidites, 1 to 10, were calculated from peak-areapercentages according to the equation ASWY=(FLP %)^(1/n) where FLP % isthe percentage full-length product in the crude chromatogram and n thenumber of synthesis cycles. ASWY ranging from of 96.5% to 97.5% wereobtained for phosphoramidites, 1 to 10. The experimental couplingefficiencies of the phosphoramidites 1 to 10, as determined using astandard spectrophotometric dimethoxytrityl assay were in completeagreement with the ASWY and were judged satisfactory to proceed with theX24, X27, X28 ribozyme library synthesis.

Example 7 Determination of Optimal Relative Concentration of a Mixtureof 2′-O-methyl-guanosine, Cytidine, Uridine and Adenosine ProvidingEqual Representation of the Four Nucleotides

[0132] A mixture N, composed of an equimolar mixture of the four2′-O-Me-nucleoside phosphoramidites (mG=2′-O-methyl guanosine;mA=2′-O-methyl adenosine; mC=2′-O-methyl cytidine; mU=2′-O-methyluridine) was used in the synthesis of a model sequence TTXXXXTTB, whereT is 2′-deoxy-thymidine and B is a 2′-deoxy-inverted abasic polystyrenesolid-support as described in Example 6. After standard deprotection(Wincott et al., supra), the crude nonamer was analyzed on ananion-exchange HPLC column (see example 1). From the HPLC analysis, anaveraged stepwise yield (ASWY) of 99.3% was calculated (see example 6)indicating that the overall coupling efficiency of the mixture N wascomparable to that of 2′-deoxythymidine. To further assess the relativeincorporation of each of the components within the mixture, N, thefull-length product TTXXXXTTB (over 94.3% at the crude stage) wasfurther purified and subjected to base composition analysis as describedherein. Purification of the FLP from the failures is desired to getaccurate base composition.

[0133] Base Composition Analysis Summary

[0134] A standard digestion/HPLC analysis was performed: To a driedsample containing 0.5 A.sub.260 units of TTXXXXTTB, 50 μl mixture,containing 1 mg of nuclease P1 (550 units/mg), 2.85 ml of 30 mM sodiumacetate and 0.3 ml of 20 mM aqueous zinc chloride, was added. Thereaction mixture was incubated at 50 degrees C. overnight. Next, 50 μlof a mixture comprising 500 μl of alkaline phosphatase (1 units/μl), 312μl of 500 mM Tris pH 7.5 and 2316 μl water was added to the reactionmixture and incubated at 37 degrees C. for 4 hours. After incubation,the samples were centrifuged to remove sediments and the supernatant wasanalyzed by HPLC on a reversed-phase C18 column equilibrated with 25 mMKH2PO4. Samples were analyzed with a 5% acetonitrile isocratic gradientfor 8 min followed by a 5% to 70% acetonitrile gradient over 8 min.

[0135] The HPLC percentage areas of the different nucleoside peaks, oncecorrected for the extinction coefficient of the individual nucleosides,are directly proportional to their molar ratios.

[0136] The results of these couplings are shown in Table III. dT2′-OMe—C 2′-OMe—U 2′-OMe-G 2′-OMe-A Nucleoside 0.1 M 0.025 M 0.025 M0.025 M 0.025 M % area 43.81 6.04 14.07 18.54 17.54 Epsilon 260 nm 88007400 10100 11800 14900 moles 0.00498 0.00082 0.00139 0.00157 0.00118equivalent 4 0.656 1.119 1.262 0.946

[0137] As can be seen in Table III, the use of an equimolar mixture ofthe four 2′-O-methyl phosphoramidites does not provide an equalincorporation of all four amidites, but favors 2′-O-methyl-U and G andincorporates 2′-O-methyl-A and C to a lower efficiency. To alleviatethis, the relative concentrations of 2′-O-methyl-A, G, U and C amiditewere adjusted using the inverse of the relative incorporation as a guideline. After several iterations, the optimized mixture providing nearlyidentical incorporation of all four amidites was obtained as shown inTable IV below. The relative representation do not exceed 12% differencebetween the most and least incorporated residue corresponding to a +/−6%deviation from equimolar incorporation. dT 2′-OMe—C 2′-OMe—U 2′-OMe-G2′-OMe-A Nucleoside 0.1 M 0.032 M 0.022 M 0.019 M 0.027 M % area 44.478.91 11.81 15.53 19.28 Epsilon 260 nm 8800 7400 10100 11800 14900 moles0.00505 0.00120 0.00117 0.00132 0.00129 equivalent 4 0.953 0.926 1.0421.024

Example 8 A Non-Competitive Coupling Method for the Preparation of theX24, X27 and N28 Ribozyme Library 5′-a _(s) c _(s) a _(s) a _(s) ag aFXGAX Gag gcg aaa gcc Gaa Agc ccu cB-3′ wherein 2′-C-allyl-uridine, 1;4′-thio-cytidine, 2; 2′-methylthiomethyl-uridine, 3;2′-methylthiomethyl-cytidine, 4; 2′-amino-uridine, 5; N3-methyl-uridine,6; 1-β-D-(ribofuranosyl)-pyrimidine-4-one, 7;1-β-D-(ribofuranosyl)-pyrimidine-2-one, 8; 1-β-D-(ribofuranosyl)-phenyl,9; and/or 6-methyl-uridine, 10 are incorporated at the X24, X27 and F28Positions through the Mix and Split Approach

[0138] The synthesis of ten different batches of 2.5 μmol scale Gag gcgaaa gcc Gaa Agc ccu cB sequence was performed on 2′-deoxy invertedabasic polystyrene solid support B on a 394 ABI DNA synthesizer (AppliedBiosystems, Foster City, Calif.). These ten aliquots were thenseparately reacted with phosphoramidite building blocks 1 to 10according to the conditions described in example 6. After completion ofthe individual incorporation of amidites 1 to 10, their couplingefficiencies were determined to be above 95% as judged by tritylmonitoring. The 10 different aliquots bearing the ten differentsequences were mixed thoroughly and divided into ten equal subsets. Eachof these aliquots were then successively reacted with ribo-A, ribo-Gamidites and one of the amidites 1 to 10. The ten aliquots werecombined, mixed and split again in 10 subsets. At that point, the 10different polystyrene aliquots, exhibiting the following sequence: X GAXGag gcg aaa gcc Gaa Agc ccu cB, were reacted again with amidites 1 to 10separately. The aliquots were not mixed, but kept separate to obtain aunique residue at the 28th position of each of the ten pools. Theribozyme synthesis was then finished independently to yield ten randomribozymes pools. Each pool comprises a 3′-terminal inverted abasicresidue B, followed by the sequence Gag gcg aaa gcc Gaa Agc ccu c,followed with one random position X in the 24th position correspondingto a mixture of amidites 1 to 10, followed by the sequence GA, followedone random position X in the 27th position corresponding to a mixture ofamidites 1 to 10, followed by a fixed monomer F (one of the amidites 1to 10) in the 28th position and finally the 5′-terminal sequencea_(s)c_(s)a_(s)a_(s)a g a. This is represented by the sequence notation5′-a_(s)c_(s)a_(s)a_(s)ag aFX GAX Gag gcg aaa gcc Gaa Agc ccu cB-3′, inwhich X are random positions and F is a unique fixed position. The totalcomplexity of such a ribozyme library was 10³ or 1,000 members separatedin 10 pools of 100 different ribozyme sequences each.

Example 9 Competitive Coupling Method (Monomer Mixing Approach) for thePreparation of the x ²⁻⁶ and X ³⁰⁻³⁵ “Binding Arms” Ribozyme Library

[0139] Synthesis of 5′-x_(s)x_(s)x xFF cuG Au G Agg ccg uua ggc cGA AAFxxx xB-3′ is described, with F being a defined2′-O-methyl-ribonucleoside chosen among 2′-O-methyl-ribo-adenosine (mA),-guanosine (mG), -cytidine (mC), -uridine (mU) and x being an equalmixture of 2′-O-methyl-ribo-adenosine, -guanosine, -cytidine, -uridine.

[0140] The syntheses of this ribozyme library was performed with an ABI394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) usingstandard nucleic acid synthesis reagents and synthesis protocols, withthe exception of an extended (7.5 min) coupling time for theribonucleoside phosphoramidites (upper case) and 2′-amino-uridinephosphoramidite, u, (2.5 min) coupling time for the2′-O-methyl-ribonucleoside phosphoramidites (lower case) and the2′-O-methyl-ribonucleoside phosphoramidites mixture, n.

[0141] Sixty four (64) batches of 0.086 g aliquots of3′-O-DMT-2′-deoxy-inverted abasic-Polystyrene (B) solid-support loadedat 29 μmol/g and equivalent to a 2.5 μmol scale synthesis wereindividually reacted with a 27:32:19:22/v:v:v:v mixture, x, ofmA:mC:mG:mU diluted in dry acetonitrile to 0.1 M as described in example2. This synthesis cycle was repeated for a total of four times. The 64aliquots were then grouped into four subsets of sixteen aliquots(Class 1) that were reacted with either mA, mG, mC, mU to synthesize then6 position. This accomplished, the sequence: 5′-cuG Au G Agg ccg uuaggc cGA AA was added onto the 6 position of the 64 aliquots constitutingClass 1. Each subset of Class 1 was then divided into four subsets offour aliquots (Class 2) that were reacted with either mA, mG, mC, mU tosynthesize the F30 position. Each subset of Class 2 was then dividedinto four subsets of one aliquot (Class 3) that were reacted with eithermA, mG, mC, mU to synthesize the F31 position. Finally, the randomsequence 5′-x_(s)x_(s)x x was added onto each of the 64 aliquots.

[0142] The ribozyme library yielded sixty four random ribozymes poolseach having an equal mixture of the four 2′-O-methyl-nucleoside at theposition x2 to 6 and x30 to 35, and a defined 2′-O-methyl-nucleosidechosen among mA, mC, mG, mU at the positions F6, F30 and F31. The totalcomplexity of such a “binding arms” ribozyme library was 4¹¹ or4,194,304 members separated in 64 pools of 65,536 different ribozymesequences each.

Example 10 Competitive Coupling Method (Monomer Mixing Approach) for thePreparation of the Position 15 to 18 “Loop II” Ribozyme Library

[0143] Synthesis of 5′ UCU CCA UCU GAU GAG GCC XXF XGG CCG AAA AUC CCU3′ is described, with F being a defined ribonucleoside chosen amongadenosine (A), guanosine (G), cytidine (C), uridine (U) and X being anequal mixture of adenosine (A), guanosine (G), cytidine (C), uridine(U).

[0144] The syntheses of this ribozyme library was performed with an ABI394 DNA synthesizer (Applied Biosystems, Foster City, Calif.) usingstandard nucleic acid synthesis reagents and synthesis protocols, withthe exception of an extended (7.5 min) coupling time for theribonucleoside phosphoramidites (A, G, C, U) and the ribonucleosidephosphoramidite mixture, X.

[0145] Four batches (4) of 2.5 μmol scale of GG CCG AAA AUC CCU sequencewere synthesized on 0.085 g samples of5′-O-DMT-2′-O-TBDMS-3′-succinyl-uridine-Polystyrene (U) solid-supportloaded at 29.8 μmol/g. To synthesize the position X15, the four aliquotsof solid-supports were individually reacted with a 30:26:24:20/v:v:v:vmixture, X, of A:C:G:U diluted in dry acetonitrile to 0.1 M according tothe optimized conditions for the DNA phosphoramidites mixed-basecoupling as described in the DNA Synthesis Course Manual published byPerkin-Elmer-Applied Biosystem Division. (DNA Synthesis Course Manual:Evaluating and isolating synthetic oligonucleotides, the complete guide,p. 2-4, Alex Andrus, August 1995). The four aliquots of solid-supportswere then individually reacted with either of the four ribonucleosidephosphoramidites (A, G, C, U) to create the F16 position. The positionX17 and X18 were then added onto the F16 (either A, G, C or U) of thefour aliquots of solid-supports by repeating twice the same procedureused for the position X15.

[0146] The synthesis of the ribozyme library was then ended by addingthe sequence 5′-UCU CCA UCU GAU GAG GCC on the position X18 of each ofthe four subsets of the ribozyme library. The ribozyme library yieldedfour random ribozymes pools that each have an equal mixture of the fourribonucleoside (A, G, C and U) at the position X15, X17 and X18, and adiscrete ribonucleoside chosen among A, C, G or U at the positions F16.The total complexity of such a loop II ribozyme library was 256 membersseparated in 4 pools of 64 different ribozyme sequences.

Example 11 Arm-Combinatorial Library Screening for Ribozyme AccessibleSites within Bcl-2, K-ras and Urokinase Plasminogen Activator (UPA)

[0147] Substrate synthesis through in vitro transcription: Run-offtranscripts for Bcl-2 and K-ras were prepared using linearized plasmids(975 and 796 nucleotides respectively). Transcripts for UPA wereproduced from a PCR generated DNA fragment containing a T7 promoter (400nucleotides). Transcription was performed using the T7 Megascripttranscription kit (Ambion, Inc.) with the following conditions: a 50 μlreaction volume containing: 7.5 mM each of ATP, CTP, UTP, and GTP, 2 mMguanosine, 5 ul 10× T7 reaction buffer, 5 ul T7 enzyme mix, and 0.5 νgof linearized plasmid or DNA template generated using PCR. The mixturewas incubated at 37° C. for 4 hours (6 hours for transcripts >500bases). Guanosine was added to the transcription reactions so that thefinal transcript could be efficiently 5′-end labeled without priorphosphatase treatment. Transcription volume was then increased to 200 μlwith buffer containing 50 mM TRIS pH 7.5, 100 mM KCl, and 2 mM MgCl₂ andspin column purified over Bio-Gel P-60 (BioRad) equilibrated in the samebuffer. 100 μl of the transcript was then applied to 750 μl of packedresin. Spin column flow-through was used directly in a 5′-end labelingreaction as follows (100 μl final volume): 82 ul of P-60 spin columnpurified transcript, 10 μl 10× polynucleotide kinase buffer, 4 ul 10U/μL Polynucleotide Kinase (Boehringer/Mannheim) and 4 μl 150 uCi/ulGamma-32P-ATP (NEN) were incubated together at 37° C. for one hour. Thereaction volume was increased to 200 μl with buffer containing 50 mMTRIS pH 7.5, 100 mM KCl and 2 mM MgCl₂ and the sample was then purifiedover Bio-Gel P-60 packed spin column as described above. Approximatespecific activities of the 5′-end labeled transcripts were determinedvia BioScan and stored frozen at −20° C.

[0148] Synthesis of Ribozyme pools: A combinatorial arm ribozymelibrary, as pictured in FIG. 23, was synthesized to identify the optimalsite in a defined mRNA target. All ribozymes within these librariescontain two binding arms, each containing 6 nucleotides. The 8 mostflanking positions, designated by X, are randomized with the four2′-O-methylribo-nucleotide residues. Position A_(15.1) is an essentialribonucleotide and is not randomized. The catalytic core/stem II/loop IIof the combinatorial ribozyme template is fixed with a chemistry thatprovides enhanced catalytic rate. Specifically, positions 4 and 7contain 2′-deoxy-2′-amino uridine (italized in figure) and positions G5,A6, G8, A9, G12, A13, A14, and A15.1 are ribose (uppercase).

[0149] All 4,194,304 (4¹¹) possible ribozymes, each containing adifferent binding arm sequence, are represented in each library. Inorder to reduce the complexity for testing of the library, 64 pools weresynthesized each having a discrete or fixed nucleotide composition atpositions 2.1, 2.2 and 15.2 (F in FIG. 23). The total complexity of thelibrary remains the same but each of the 64 subsets is comprised of 4⁸(65,536) ribozymes that differ in the 8 “x” positions. The randomizedpositions were synthesized by the phosphoramidite pooling protocol(supra). The molar ratio of 2′-O-methyl phosphoramidites used was thefollowing: 32% 2′-O-Methyl-C; 22% 2′-O-Methyl-U, 29% 2′-O-Methyl-G, and27% 2′-O-Methyl-A.

[0150] In vitro ribozyme-transcript cleavage reactions: Cleavagereactions were carried out as follows: 5′-end labeled transcript(˜2-4×10⁴ dpm/ul final) was incubated with 10 μM ribozyme pool in 50 mMTRIS pH 7.5, 50 mM NaCl, 2 mM MgCl₂ and 0.01 % SDS for 24-48 hours atroom temperature (˜22° C.). An equal volume of gel loading dye (95%formamide, 0.01M EDTA, 0.0375% bromophenol blue, and 0.0375% xylenecyanol) was added to stop the reaction and the samples are heated to 95°C. Reactions (1-2×10⁵ dpm per lane) were run on a 5% denaturingpolyacrylamide gel containing 7M urea and 1× TBE. Gels are dried andimaged using the PhosphorImager system (Molecular Dynamics). Ambion,Inc. RNA Century Marker Plus RNA standards body labeled in a T7Megascript reaction as described above using 3 μl of 10 mCi/mlAlpha-³²P-ATP (BioRad) and 0.5 μg Century RNA template and subsequentlyspin column purified over Bio-Gel P-6 (Bio-Rad) were used as a sizereference on the gel. Cleavage product sizes were determined using theRNA standards which provided an approximate site of cleavage (est. Sizein Figure). Because each of the ribozyme pools has three positionswithin the binding arms fixed, it is possible to identify all of thepotential ribozyme sites that can potentially be cleaved by that pool.The estimated size of the cleavage product is therefore compared withthe potential sites to identify the exact site of cleavage.

[0151] The screening method identified 13 sites for ribozyme activity(FIG. 19) on the bcl-2 transcript, 15 sites on the K-ras transcript(FIG. 20), and 7 sites (FIG. 21) on the UPA transcript.

Example 12 Reduction of Bcl-2 mRNA using Optimized Ribozymes

[0152] Two ribozymes targeted against the same site in the bcl-2transcript (Seq.ID No.9) were synthesized, but the two ribozymes werestabilized using two different chemistries (U4/U7 2′-amino and U42′-C-allyl). MCF-7 cells were treated in serum delpleted media for 7days prior to treatment with ribozymes. Ribozymes (200 nM) weredelivered using lipofectamine (7.2 mM) for 3 hours into these cells at50% confluency. Cellular RNA was harvested 24 hours after delivery,analyzed by RNase protectection analysis (RPA) and normalized to GAPDHmRNA in triplicate samples. Both ribozymes gave a reduction in bcl-2mRNA (FIG. 22). A ribozyme targeted against an irrelevant mRNA (c-myb)had no effect on the ratio of bcl-2 mRNA to GAPDH mRNA. All reduction ofbcl-2 RNA was statistically significant with respect to untreatedsamples and samples treated with the irrelevant ribozyme.

[0153] Diagnostic Uses

[0154] Enzymatic nucleic acids of this invention may be used asdiagnostic tools to examine genetic drift and mutations within diseasedcells or to detect the presence of target RNA in a cell. The closerelationship between ribozyme activity and the structure of the targetRNA allows the detection of mutations in any region of the moleculewhich alters the base-pairing and three-dimensional structure of thetarget RNA. By using multiple ribozymes described in this invention, onemay map nucleotide changes which are important to RNA structure andfunction in vitro, as well as in cells and tissues. Cleavage of targetRNAs with ribozymes may be used to inhibit gene expression and definethe role (essentially) of specified gene products in the progression ofdisease. In this manner, other genetic targets may be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combinational therapies (e.g., multiple ribozymes targeted todifferent genes, ribozymes coupled with known small molecule inhibitors,or intermittent treatment with combinations of ribozymes and/or otherchemical or biological molecules). Other in vitro uses of ribozymes ofthis invention are well known in the art, and include detection of thepresence of mRNAs associated with disease condition. Such RNA isdetected by determining the presence of a cleavage product aftertreatment with a ribozyme using standard methodology.

[0155] In a specific example, ribozymes which can cleave only wild-typeor mutant forms of the target RNA are used for the assay. The firstribozyme is used to identify wild-type RNA present in the sample and thesecond ribozyme will be used to identify mutant RNA in the sample. Asreaction controls, synthetic substrates of both wild-type and mutant RNAwill be cleaved by both ribozymes to demonstrate the relative ribozymeefficiencies in the reactions and the absence of cleavage of the“non-targeted” RNA species. The cleavage products from the syntheticsubstrates will also serve to generate size markers for the analysis ofwild-type and mutant RNAs in the sample population. Thus each analysiswill require two ribozymes, two substrates and one unknown sample whichwill be combined into six reactions. The presence of cleavage productswill be determined using an RNAse protection assay so that full-lengthand cleavage fragments of each RNA can be analyzed in one lane of apolyacrylamide gel. It is not absolutely required to quantify theresults to gain insight into the expression of mutant RNAs and putativerisk of the desired phenotypic changes in target cells. The expressionof mRNA whose protein product is implicated in the development of thephenotype is adequate to establish risk. If probes of comparablespecific activity are used for both transcripts, then a qualitativecomparison of RNA levels will be adequate and will decrease the cost ofthe initial diagnosis. Higher mutant form to wild-type ratios will becorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

[0156] Additional Uses

[0157] Potential usefulness of sequence-specific enzymatic nucleic acidmolecules of the instant invention might have many of the sameapplications for the study of RNA that DNA restriction endonucleaseshave for the study of DNA (Nathans et al., 1975 Ann. Rev. Biochem.44:273). For example, the pattern of restriction fragments could be usedto establish sequence relationships between two related RNAs, and largeRNAs could be specifically cleaved to fragments of a size more usefulfor study. The ability to engineer sequence specificity of the ribozymeis ideal for cleavage of RNAs of unknown sequence.

[0158] Other embodiments are within the following claims. TABLE ICharacteristics of naturally occurring ribozymes Group I Introns Size:˜150 to >1000 nucleotides. Requires a U in the target sequenceimmediately 5′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site. Reaction mechanism: attack by the 3′-OH ofguanosine to generate cleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintainance of the active structure. Over 300 known members of thisclass. Found as an intervening sequence in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. Major structural features largely established throughphylogenetic comparisons, mutagenesis, and biochemical studies [^(i,)^(ii)]. Complete kinetic framework established for one ribozyme [^(iii,)^(iv,) ^(v,) ^(vi)]. Studies of ribozyme folding and substrate dockingunderway [^(vii,) ^(viii,) ^(ix)]. Chemical modification investigationof important residues well established [^(x,) ^(xi)]. The small (4-6 nt)binding site may make this ribozyme too non-specific for targeted RNAcleavage, however, the Tetrahymena group I intron has been used torepair a “defective” β-galactosidase message by the ligation of newβ-galactosidase sequences onto the defective message [^(xii)]. RNAse PRNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portion of a ubiquitousribonucleoprotein enzyme. Cleaves tRNA precursors to form mature tRNA[^(xiii)]. Reaction mechanism: possible attack by M²⁺-OH to generatecleavage products with 3′-OH and 5′-phosphate. RNAse P is foundthroughout the prokaryotes and eukaryotes. The RNA subunit has beensequenced from bacteria, yeast, rodents, and primates. Recruitment ofendogenous RNAse P for therapeutic applications is possible throughhybridization of an External Guide Sequence (EGS) to the target RNA[^(xiv,) ^(xv)] Important phosphate and 2′ OH contacts recentlyidentified [^(xvi,) ^(xvii)] Group II Introns Size: >1000 nucleotides.Trans cleavage of target RNAs recently demonstrated [^(xviii,) ^(xix)].Sequence requirements not fully determined. Reaction mechanism: 2′-OH ofan internal adenosine generates cleavage products with 3′-OH and a“lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Only naturalribozyme with demonstrated participation in DNA cleavage [^(xx,) ^(xxi)]in addition to RNA cleavage and ligation. Major structural featureslargely established through phylogenetic comparisons [^(xxii)].Important 2′ OH contacts beginning to be identified [^(xxiii)] Kineticframework under development [^(xxiv)] Neurospora VS RNA Size: ˜144nucleotides. Trans cleavage of hairpin target RNAs recently demonstrated[^(xxv)]. Sequence requirements not fully determined. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. Binding sites andstructural requirements not fully determined. Only 1 known member ofthis class. Found in Neurospora VS RNA. Hammerhead Ribozyme (see textfor references) Size: ˜13 to 40 nucleotides. Requires the targetsequence UH immediately 5′ of the cleavage site. Binds a variable numbernucleotides on both sides of the cleavage site. Reaction mechanism:attack by 2′-OH 5′ to the scissile bond to generate cleavage productswith 2′,3′-cyclic phosphate and 5′-OH ends. 14 known members of thisclass. Found in a number of plant pathogens (virusoids) that use RNA asthe infectious agent. Essential structural features largely defined,including 2 crystal structures [^(xxvi,) ^(xxvii)] Minimal ligationactivity demonstrated (for engineering through in vitro selection)[^(xxviii)] Complete kinetic framework established for two or moreribozymes [^(xxix)]. Chemical modification investigation of importantresidues well established [^(xxx)]. Hairpin Ribozyme Size: ˜50nucleotides. Requires the target sequence GUC immediately 3′ of thecleavage site. Binds 4-6 nucleotides at the 5′-side of the cleavage siteand a variable number to the 3′- side of the cleavage site. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. 3 known members ofthis class. Found in three plant pathogen (satellite RNAs of the tobaccoringspot virus, arabis mosaic virus and chicory yellow mottle virus)which uses RNA as the infectious agent. Essential structural featureslargely defined [^(xxxi,) ^(xxxii,) ^(xxxiii,) ^(xxxiv)] Ligationactivity (in addition to cleavage activity) makes ribozyme amenable toengineering through in vitro selection [^(xxxv)] Complete kineticframework established for one ribozyme [^(xxxvi)]. Chemical modificationinvestigation of important residues begun [^(xxxvii,) ^(xxxviii)]Hepatitis Delta Virus (HDV) Ribozyme Size: ˜60 nucleotides. Transcleavage of target RNAs demonstrated [^(xxxix)]. Binding sites andstructural requirements not fully determined, although no sequences 5′of cleavage site are required. Folded ribozyme contains a pseudoknotstructure [^(xl)]. Reaction mechanism: attack by 2′-OH 5′ to thescissile bond to generate cleavage products with 2′,3′-cyclic phosphateand 5′-OH ends. Only 2 known members of this class. Found in human HDV.Circular form of HDV is active and shows increased nuclease stability[^(xli)]

[0159] TABLE II 2.5 μmol RNA Synthesis Cycle Wait Reagent EquivalentsAmount Time* Phosphoramidites 6.5 163 μL 2.5 S-Ethyl Tetrazole 23.8 238μL 2.5 Acetic Anhydride 100 233 μL   5 sec N-Methyl Imidazole 186 233 μL  5 sec T C A 83.2 1.73 ml  21 sec Iodine 8.0 1.18 ml  45 secAcetonitrile NA 6.67 ml NA

[0160]

1 52 1 13 DNA Artificial Sequence misc_feature Accessible site withinBcl-2 transcript 1 ttgcttttcc tct 13 2 13 DNA Artificial Sequencemisc_feature Accessible site within Bcl-2 transcript 2 gttgcttttc ctc 133 13 DNA Artificial Sequence misc_feature Accessible site within Bcl-2transcript 3 gtgcctatct gag 13 4 13 DNA Artificial Sequence misc_featureAccessible site within Bcl-2 transcript 4 gctcctctag act 13 5 13 DNAArtificial Sequence misc_feature Accessible site within Bcl-2 transcript5 cgcccttcac cgc 13 6 13 DNA Artificial Sequence misc_feature Accessiblesite within Bcl-2 transcript 6 agctcttcag gga 13 7 13 DNA ArtificialSequence misc_feature Accessible site within Bcl-2 transcript 7tcctctagac tcg 13 8 13 DNA Artificial Sequence misc_feature Accessiblesite within Bcl-2 transcript 8 ctgagtacct gaa 13 9 13 DNA ArtificialSequence misc_feature Accessible site within Bcl-2 transcript 9ttgagttcgg tgg 13 10 13 DNA Artificial Sequence misc_feature Accessiblesite within Bcl-2 transcript 10 tgaagtacat cca 13 11 13 DNA ArtificialSequence misc_feature Accessible site within Bcl-2 transcript 11tgtggtccac ctg 13 12 13 DNA Artificial Sequence misc_feature Accessiblesite within Bcl-2 transcript 12 ccccatccag ccg 13 13 13 DNA ArtificialSequence misc_feature Accessible site within Bcl-2 transcript 13ctggatccag gat 13 14 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 14 caggctcagg agt 13 15 13 DNA ArtificialSequence misc_feature Accessible site within Kras transcript 15aatactaaat cat 13 16 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 16 ttgtgtattt gcc 13 17 13 DNA ArtificialSequence misc_feature Accessible site within Kras transcript 17aggagtacag tgc 13 18 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 18 tgtggtagtt gga 13 19 13 DNA ArtificialSequence misc_feature Accessible site within Kras transcript 19ggtagttgga gct 13 20 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 20 gggtgttgac gat 13 21 13 DNA ArtificialSequence misc_feature Accessible site within Kras transcript 21aggagttatg ggc 13 22 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 22 gcaggtcaag agg 13 23 13 DNA ArtificialSequence misc_feature Accessible site within Kras transcript 23aagagtaaag gac 13 24 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 24 gtgtatttgc cat 13 25 13 DNA ArtificialSequence misc_feature Accessible site within Kras transcript 25agatattcac cat 13 26 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 26 cattatagag aac 13 27 13 DNA ArtificialSequence misc_feature Accessible site within Kras transcript 27attcattgag acc 13 28 13 DNA Artificial Sequence misc_feature Accessiblesite within Kras transcript 28 caccattata gag 13 29 13 DNA ArtificialSequence misc_feature Accessible site within UPA transcript 29gtcactttta ccg 13 30 13 DNA Artificial Sequence misc_feature Accessiblesite within UPA transcript 30 gccgcttgtc caa 13 31 13 DNA ArtificialSequence misc_feature Accessible site within UPA transcript 31gggcctaaag ccg 13 32 13 DNA Artificial Sequence misc_feature Accessiblesite within UPA transcript 32 cactgtcctt cag 13 33 13 DNA ArtificialSequence misc_feature Accessible site within UPA transcript 33gcttgtccaa gag 13 34 13 DNA Artificial Sequence misc_feature Accessiblesite within UPA transcript 34 ggccatctac agg 13 35 13 DNA ArtificialSequence misc_feature Accessible site within UPA transcript 35caccatcgag aac 13 36 11 RNA Artificial Sequence Miscellaneous targetsequence 36 nnnnuhnnnn n 11 37 28 RNA Artificial Sequence EnzymaticNucleic Acid 37 nnnnncugan gagnnnnnnc gaaannnn 28 38 49 RNA ArtificialSequence Enzymatic Nucleic Acid 38 cuccaccucc ucgcggunnn nnnngggcuacuucgguagg cuaagggag 49 39 176 RNA Artificial Sequence Enzymatic NucleicAcid 39 gggaaagcuu gcgaagggcg ucgucgcccc gagcgguagu aagcagggaacucaccucca 60 auuucaguac ugaaauuguc guagcaguug acuacuguua ugugauugguagaggcuaag 120 ugacgguauu ggcguaaguc aguauugcag cacagcacaa gcccgcuugcgagaau 176 40 36 RNA Artificial Sequence Enzymatic Nucleic Acid 40acaaagacug augaggccga aaggccgaaa gcccuc 36 41 36 RNA Artificial SequenceEnzymatic Nucleic Acid 41 acaaagauug augaggccga aaggccgaaa gcccuc 36 4236 RNA Artificial Sequence Enzymatic Nucleic Acid 42 acaaagaucgaugaggccga aaggccgaaa gcccuc 36 43 36 RNA Artificial Sequence EnzymaticNucleic Acid 43 acaaagaucg augaggccga aaggccgaaa gcccuc 36 44 36 RNAArtificial Sequence Enzymatic Nucleic Acid 44 acaaagaucg angaggccgaaaggccgaaa gcccuc 36 45 36 RNA Artificial Sequence Enzymatic NucleicAcid 45 ucuccaucug augaggccga aaggccgaaa aucccu 36 46 17 RNA ArtificialSequence Target sequence 46 cagggauuaa uggagau 17 47 37 RNA ArtificialSequence Enzymatic Nucleic Acid 47 ucuccaucug augaggccga aaggccgaaaaucccuu 37 48 37 RNA Artificial Sequence Enzymatic Nucleic Acid 48ucuccaucug augaggccgu uaggccgaaa aucccuu 37 49 34 RNA ArtificialSequence Enzymatic Nucleic Acid 49 caaagacuga ugaggccgaa aggccgaaag cccu34 50 34 RNA Artificial Sequence misc_feature (1)..(4) The letter “n”stands for any base. 50 nnnnnncuga ugaggccguu aggccgaaan nnnn 34 51 47RNA Artificial Sequence Enzymatic Nucleic Acid 51 nnnngaagnn nnnnnnnnnaaahannnnnn nacauuacnn nnnnnnn 47 52 15 RNA Artificial SequenceMiscellaneous target sequence 52 nnnnnnnyng hynnn 15

1. A method for identifying variants of a nucleic acid catalystcomprising the steps of: a) selecting at least three positions withinsaid nucleic acid catalyst to be varied with a predetermined group ofdifferent nucleotides; b) synthesizing a first class of different poolsof said nucleic acid catalyst, wherein the number of pools synthesizedis equal to the number of nucleotides in the predetermined group ofdifferent nucleotides, wherein at least one of the positions to bevaried in each pool comprises a defined nucleotide selected from thepredetermined group of different nucleotides and the remaining positionsto be varied comprise a random mixture of nucleotides selected from thepredetermined group of different nucleotides; c) testing the differentpools of said nucleic acid catalyst under conditions suitable for saidpools to show a desired attribute and identifying the pool with saiddesired attribute and wherein the position with the defined nucleotidein the pool with the desired attribute is made constant in subsequentsteps; d) synthesizing a second class of different pools of nucleic acidcatalyst, wherein at least one of the positions to be varied in each ofthe second class of different pools comprises a defined nucleotideselected from the predetermined group of different nucleotides and theremaining positions to be varied comprise a random mixture ofnucleotides selected from the predetermined group of differentnucleotides; e) testing the second class of different pools of saidnucleic acid catalyst under conditions suitable for showing (a) desiredattribute and identifying the pool with said desired attribute andwherein the position with the defined nucleotide in the pool with thedesired attribute is made constant in subsequent steps; and f) repeatingthe process similar to steps d and e until every position selected insaid nucleic acid catalyst to be varied is made constant.
 2. A methodfor identifying novel nucleic acid molecules in a biological system,comprising the steps of: a) synthesizing a pool of nucleic acidcatalyst(s) having a substrate binding domain and a catalytic domain,wherein said substrate binding domain comprises a random sequence; b)testing the pools of nucleic acid catalyst under conditions suitable forshowing a desired effect in said biological system and identifying thenucleic acid catalyst showing said desired effect; c) using anoligonucleotide comprising the sequence of the substrate binding domainof the nucleic acid catalyst showing said desired effect as a probe toscreening said biological system for nucleic acid moleculescomplementary to said probe; and d) isolating and sequencing saidcomplementary nucleic acid molecules.